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Sep 2019

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Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
制备果蝇多线染色体并通过多重荧光关联对染色质结构进行免疫荧光分析   

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Abstract

Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest.

Keywords: Polytene chromosome squashes (多线染色体按压), Chromatin structure (染色质结构), Protein recruitment (蛋白募集), Pearson correlation coefficients (Pearson 相关系数), Immunofluorescence analysis (免疫荧光分析)

Background

Polytene chromosomes from Drosophila larval salivary glands have an unusually large size (visible under a typical cell-culture microscope). This is due to altered cell cycle processes that result in ~1,000 copies of each chromosome that do not segregate from each other. The resulting large chromosomes have stereotyped and reproducible alternating bands of condensed and decondensed chromatin, as chromosomes are aligned and locus-specific chromatin structure is visible upon polytenization. The nature of these chromosomes makes the analysis of chromatin structure uniquely visible relative to diploid cell types, enabling analysis of chromatin changes throughout development, transcriptional activation, or upon perturbation. Additionally, genome-wide and site-specific chromatin binding of proteins can be visualized by immunofluorescence staining of polytene chromosomes (Paro, 2008; Cai et al., 2010), allowing for investigation of the relationships between candidate proteins and chromatin condensation, transcriptional activity, and histone modifications. In this manner, polytene chromosome staining provides a rapid and inexpensive alternative or supplement to molecular assays such as Chromatin Immunoprecipitation, coupled to sequencing (ChIP-seq).

Our squashing and fluorescence analysis pipeline was specifically designed for analysis of chromatin structure and protein recruitment/changes at single bands of interest, originally for a band of LacI-tethered protein bound to a genomic lacO repeat array (Kuhn et al., 2019). In this study, we targeted specific nuclear pore components, fused to LacI, to lacO arrays, integrated at defined sites in the genome. We then aimed to determine whether such targeting leads to recruitment of candidate interacting partners, and also to changes in target chromatin compaction. This approach involved identification of a single band of interest (harboring the lacO site) within an entire genome of spread chromosomes, which required high-quality chromosome spreading for increased visibility and analysis of chromosome banding patterns. Additionally, screening through multiple antibodies to assay for many candidate interacting partners required an extremely reproducible squashing protocol, with minimal sample failure rate. To achieve these goals, we developed this optimized polytene squashing protocol, which can be utilized to improve image quality and sample success rate. It is also especially useful for chromosome ‘mappability’ (determining genomic location by cytological mapping of polytene banding patterns) and for more high-throughput applications.

In order to reliably analyze site-specific changes in chromatin structure (i.e., changes in chromatin condensation as assessed by DNA stain fluorescence intensity), and in protein recruitment, we developed a semi-automated MATLAB function. This function, PCCcalc, calculates the Pearson Correlation Coefficients (PCC) between multiple fluorescence signals at a defined genomic site on a pixel-by-pixel basis in an unbiased manner. This greatly reduces data analysis time and subjectivity derived from manual analysis and user-based variability. Additionally, this approach circumvents the impact of slide-to-slide variability in fluorescence background or image acquisition, as the analysis focuses on the relationship between fluorescence signals more than absolute values of a given signal. This method can be used on any easily identifiable site on polytene chromosomes to determine binding correlations between multiple protein factors, or to find correlations between protein of interests and associated changes in chromatin compaction. Furthermore, we believe this analysis method can be extended to determine fluorescence correlation beyond the described use in polytene chromosomes, and can be used to analyze co-localization or co-occurrence of fluorescently labeled biological markers in many other contexts and cell types.

Materials and Reagents

  1. Filter paper (Fisherbrand, catalog number: 05-714-4 )
  2. Hammer with a rubber head (Graham Field, catalog number: 1312-1 )
  3. Fine sharpie
  4. 1.5 ml tubes (USA Scientific, catalog number: 1615-5510 )
  5. Dissection Well Dish/Pyrex Spot Plate (Fisher Scientific, catalog number: 13-748B )
  6. Dumont Fine #55 Forceps (Fine Sceince Tools, catalog number: 11295-51 )
  7. Coverslips (Fisherbrand, catalog number: 12-542A )
  8. Coplin Jar (VWR, catalog number: 74830-150 )
  9. Humidity chamber (such as Pyrex Lidded Glass Baking Dish)
  10. Poly-L-Lysine-treated Slides (Polysciences, catalog number: 22247 )
  11. Poly-L-Lysine (alternative to pre-treated slides above) (Sigma-Aldrich, catalog number: P8920-100 ml )
  12. Sigmacote (Sigma-Aldrich, catalog number: SL2-100ML )
  13. Phosphate Buffered Saline (PBS) reagents:
    1. NaCl (Fisher, catalog number: BP358-212 )
    2. KCl (Fisher, catalog number: BP366-500 )
    3. Na2HPO4 (Sigma, catalog number: S3264-500G )
    4. KH2PO4 (Sigma, catalog number: P9791-1KG )
  14. Primary Antibodies used: anti-LacI (Rockland, catalog number: 600-401-B05 ), anti-Nup98 (Capelson et al., 2010), anti-Brm (Nakayama et al., 2012)
  15. Tween 20 (Fisher, catalog number: BP337-500 )
  16. Glacial Acetic Acid (Fisher, catalog number: BP2401-212 )
  17. 16% Paraformaldehyde (PFA) (Alfa Aesar, catalog number: 43368 )
  18. Liquid nitrogen
  19. Kimwipes or other tissue-style wiper (Fisher Scientific, catalog number: 06-666A )
  20. Fluorescent Secondary Antibodies (e.g., Thermo Fisher, catalog number: A-11004 )
  21. Hoechst 33342 (Thermo Fisher, catalog number: H3570 ) (10 mg/ml stock)
  22. Prolong Gold Antifade (Thermo Fisher, catalog number: P36930 )
  23. PBS (see Recipes)
  24. PBST (see Recipes)
  25. Fixation solution (see Recipes)
  26. 45% acetic acid (see Recipes)
  27. Blocking solution/Antibody cocktail base (see Recipes)

Equipment

  1. Forceps
  2. Pipettes
  3. Chemical hood
  4. Dissecting Microscope (such as Leica, model: Leica S6E )
  5. Cell Culture/Phase Contrast Microscope (such as Leica, model: Leica DM IL )
  6. Widefield epifluorescent microscope (such as Leica, model: Leica DM6000 ) and/or laser scanning confocal microscope
  7. (Optional) Orbital shaker (VWR, catalog number:12620-938)

Software

  1. MATLAB (license) (necessary for analysis)
  2. Graphing/Statistical Analysis software, e.g., Excel, Prism (licenses) (necessary for analysis)
  3. (Optional) ImageJ (free)

Procedure

  1. Prior preparation of Sigmacote treated coverslips and poly-L-lysine treated slides
    Note: Coverslips are treated with Sigmacote to prevent sticking of chromosomes and to encourage preferential adherence of chromosomes to poly-L-lysine treated slides for subsequent staining and imaging. These are best if treated prior to the day of the experiment.

    Sigmacote treated coverslips
    1. Wrap a 1.5 ml tube rack (or similar structure) in plastic wrap and set on a larger rectangle of paper towels in a chemical hood, see Figure 1.
    2. Pour Sigmacote (stored at 4 °C) into the Sigmacote bottle’s cap.
    3. Using clean, fine forceps, dip the coverslips individually into Sigmacote, completely submerging. This is best with curved/bent tips so that the forceps are minimally touching the coverslip (see Step B7 below).
    4. Promptly remove the coverslip, touch the corner of the coverslip to the surface of the liquid in the Sigmacote bottle’s cap to remove excess, and place the coverslip at ~60° angle against the plastic-covered rack on top of the paper towel so any remaining Sigmacote wicks away from coverslips (see Figure 1).
    5. Once the desired number of coverslips is made, return the Sigmacote from the cap into the Sigmacote bottle, store at 4 °C, use within 1 year.
    6. Leave coverslips to dry overnight in a chemical hood (can use after ~1 h if time is limited).
    7. Store coverslips in 4 °C (stacked back in the original container is fine after overnight drying), best if used within ~2 months.


      Figure 1. A simple lab set-up for drying coverslips after Sigmacote treatment. Coverslips to be used for polytene chromosome squashes are treated with Sigmacote and placed leaning against a 1.5 ml tube rack wrapped in plastic wrap, placed on paper towels, inside of the chemical hood, as described in A1-A4 above.

    Poly-L-lysine treated slides
    Poly-L-lysine slides can be purchased pretreated (we found pretreated slides to have lower autofluorescence background compared to self-treated slides), but to save costs, or if pre-treated slides are unavailable, standard frosted microscope slides can be treated with poly-L-lysine similarly to treating coverslips with Sigmacote:
    1. Pour poly-L-lysine into a glass Coplin jar.
    2. Insert frosted microscope slides so that the non-frosted glass part of the slide (i.e., excluding frosted label area) is submerged in the Poly-L-lysine.
    3. Once the Coplin jar is filled with slides, remove the slides in original order in which they were treated, and place at a ~60° angle against a vertical surface (such as conical rack) on paper towels.
    4. Let slides dry overnight, minimum ~2 h if in a rush.
    5. Store at 4 °C (stacked back in original slide box is fine after overnight drying), best if used within ~2 months.

  2. Gland Dissection and Chromosome squashing
    Before beginning dissections, gather all materials, make fixation solutions, and fill an appropriate container with liquid nitrogen.
    1. Pick ideal 3rd instar wandering larvae and place up to 4 in a glass dissection well dish in PBS.

      Note: Picking larvae is the most important step for getting highest quality squashes! The 3rd instar wandering larvae that you use for harvesting salivary glands should be:
      1. From un-crowded vials/bottles–ideal larvae have had low competition for nutrients and are fat and healthy.
      2. Late wandering 3rd instar, minimally moving, but not yet in pre-pupa stage, as older larvae (primarily moving only mouth) will have larger chromosomes and give the best squashes.
        1)
        Larvae still quickly moving around vial/bottle walls will be younger and have had fewer polytene chromosome replications and therefore smaller chromosomes.
        2)
        Larvae too close to pupation/pre-pupa stage will however have too much salivary gland "glue" protein production, and this can lead to difficult spreading and increased immunofluorescence background. To avoid this, large "glassy" or more transparent salivary glands (which are full of the glue-like substance that releases upon puncturing), (see Figure 2) should be skipped for best quality squashes. Never take larvae with anterior spiracles extended, as they will almost always have these types of glands.
      3. From the very first wave of wandering larvae that usually show up 6-7 days after the vial/cross has been set up or flipped (if reared at 21-22 °C room temperature).
        Quality squashes can come from larvae taken on the second day of wandering in uncrowded conditions, but salivary glands and chromosomes are generally largest from larvae from the very first wave, so success rate/quality will be highest from first-wave larvae.
      4. Females (unless interested in sex-specific differences). Female larvae tend to have larger salivary glands and polytene chromosomes than male larvae. Female sex of 3rd instar larvae is determined by the absence of large, transparent, spherical gonads visible on either side of the interior of larvae, about 1/3 up from the posterior tip of the larvae. These large gonads are instead indicative of males.


        Figure 2. Different types of salivary glands isolated from 3rd instar wandering larvae. A. Typical set of salivary glands, usually opaque/“milky” in appearance, with well-defined cells visible. B. Mature glands to be avoided, taken from larva close to pupation, appearing much more clear/transparent/”glassy” due to increased glue substance, usually with less visible cellular borders. White pieces attached to glands are remaining fat body.

    2. Transfer one larva into a new well with PBS for dissection. Replace the PBS from this well after every 2-3 dissections.
    3. Dissect out salivary glands by holding larval mouth hooks with one pair of forceps and gently pulling larva in the other direction from about halfway down their body with another pair of forceps. (For demonstration of salivary gland dissections from Drosophila larvae, there are a number of public videos available, available here)
    4. Peel off the fat bodies (white strips of tissue) from the sides of glands, as this can contribute to fluorescence background in downstream imaging; however don’t spend more than a couple of minutes removing these if they are persistent, it is better to proceed to fixation sooner. Dissections should not proceed longer than ~5 min before fixation.
    5. Transfer the glands to a different dissection dish well containing ~100-200 μl of freshly made fixation solution and leave for precisely 1 min.
      1. Glands may be held by the “wishbone”/stem area where they are adjoined to transfer to fixative solution, as this prevents damaging gland cells.
      2. Transferring glands here can be made much easier by the use of a pair of forceps with one bent/curved tip, described in Step B7 below.
      3. Acetic acid serves here to disrupt nuclear membranes and aid in chromosome spreading, however not all antibodies work well under these conditions, and some may require optimization in different fixation lengths or solutions, see Note in Section C.
      Notes:
      1. In our hands, all acetic acid solutions must be made fresh every 30-45 min from 100% acetic acid to ensure proper chromosome spreading, as acetic acid dilutions go bad quickly and will make chromosomes much more difficult to spread.
      2. Additionally, once opened, it is critical that 16% PFA is stored at 4 °C (generally transferred from glass ampule to 15 ml conical tube) and ONLY used for 3 weeks for optimal chromosome spreading and structure preservation.
    6. Important! From this point forward, work quickly, yet carefully, as fixation will continue until slide is frozen in liquid nitrogen (below). Best to prepare the coverslip and label the slide during the fixation incubation above.
    7. Wipe any residue from the Sigmacote-treated coverslip using a kimwipe or other tissue-style wiper, add 7 μl of fresh 45% acetic acid onto the coverslip, and gently transfer the fragile glands into the drop of acetic acid.
      1. Glands become incredibly fragile after fixation and will require finesse to transfer.
      2. Highly recommended for this step is a pair of forceps with one bent tip, such that it forms a curved or hooked shape at the end facing toward the other tip, preventing complete closure of the forceps. These can pick up the glands in a small drop of liquid at the tip, and prevents glands from being squished and sticking to the forceps. These can be used for all transferring steps, but are especially necessary after fixation when glands become fragile.
    8. Lower the poly-L-lysine treated slide gently onto the drop of acetic acid so that the glands end up approximately in the middle of the slide.
    9. Flip over the slide and, using a gloved index finger, apply pressure on the coverslip to simultaneously push the coverslip “into” the slide while also moving the coverslip around in a circle, ~1 cm in each direction in a clockwise motion, 3 times around, to break apart the tissue and spread the cells and chromosomes (see Video 1 for Steps B9-B14).
      1. It is important to always go in a circle in one direction to prevent chromosome tangling.
      2. The coverslip should slide relatively smoothly and not get stuck. If sticking occurs, see the following Note.
      3. Optimization and practice will be required at this step for each individual to learn the appropriate movements and amount of finger-pressure to get their best spreads. Figure 3A shows an example of what chromosomes look like when more pressure is needed from this step.
      Note: The goal of moving the coverslip around is to sufficiently separate cells so they are not overlapping, to break membranes, and to spread chromosomes, but within a small enough area that all chromosomes can still be covered by a coverslip later for mounting and imaging. If the coverslip is not sliding smoothly during spreading, this will lead to chromosome tearing/stretching (Figure 3B). However, too little resistance and the cells will not break and the chromosomes will not spread enough (Figure 3A), which may be caused by too much liquid between coverslip and slide.
      Things that may help prevent coverslip sticking:
      1. Avoid clear, “gluey” glands (as discussed above in Step B1, these appear glass-like/transparent) (see Figure 2).
      2. Use freshly made, completely dry (best overnight), and fully wiped-off Sigmacote-treated coverslips, made with fresh Sigmacote (the same applies to poly-L-lysine treated slides if you are treating them yourself).
      3. Use fresh acetic acid solutions made within 30-45 min of use.

        Video 1. Polytene Chromosome Squash

    10. Flip the slide over so the coverslip is on the bottom and carefully insert the slide into a folded piece of thick filter paper.
    11. Hold two fingers on either side of where the coverslip is located to secure the slide and prevent lateral movement between slide and coverslip.
    12. Tap the coverslip area with a rubber hammer with medium force ~30 times. This step will take practice and the amount of force used will need to be optimized for each individual.
    13. Push straight down on the slide with two thumbs firmly on either side of the coverslip, without letting the coverslip slip relative to the slide (see Video 1), in order to flatten the chromosomes and prevent 3D/out-of-focus chromosome structure when imaging (Figures 3A and 3C).
    14. Remove the slide out from filter paper, and mark the location of the corners of the coverslip on the back of the slide with fine sharpie in order to locate the chromosome area in future antibody incubations and mounting.
    15. Check chromosome spreading quality under a cell culture/phase contrast microscope, under 20x or 40x objective. If spreading is satisfactory (Figures 3D-3E), proceed. Otherwise, throw the slide away and repeat with the next larva, making sure to change out PBS in the dissection well after every 2-3 larvae.

      Note: Quality control of chromosome spreads:
      1. A good chromosome spread should have chromosomes that fan out, are somewhat separated, are intact and not stretched/torn, and are flat (Figures 3D-E).
      2. If slides show most of the chromosomes torn/stretched (Figure 3B), chromosomes very tangled together (Figures A-B), or nuclei not adequately broken with chromosomes still in a tight ball (Figure 3A), it is usually very difficult to get useable data from these and it is most often a waste of time and resources to proceed with substandard slides.
      3. With this protocol, once one has practiced, we generally have a much higher success rate than with previously attempted protocols, up to 80-90% for experienced individuals starting with ideal larvae. However, you may still throw several slides away at this stage. Because of the innate variability in squashing and staining, you want to end up with a minimum of 3 quality slides per condition (genotype/antibody/etc.).


      Figure 3. Examples of poor and high quality polytene chromosome squashes. A. Under-spread squash, evident by the lack of chromosomes spreading out from the original round nuclear shape, and under-flattened squash, evident by chromosomes present in multiple focal planes, i.e., blurry and in-focus chromosomes visible simultaneously. Common fixes: To increase chromosome spreading, more pressure must be used during coverslip movement in Step B9; to decrease “blurriness,” make sure to push the coverslip into the slide with more pressure in Step B13 (avoiding lateral movement of coverslip relative to slide). Scale bar = 10 μm. Chromosomes are stained with Hoechst (shown as white), in A-E. B. Over-stretched chromosomes, visible by thin strands with poorly defined chromatin bands. Chromatin is also broken apart in places, without bands appearing in their stereotyped, organized order. Common fix: Hit the slide less forcefully with the hammer in Step B12, and also make sure to avoid any lateral movement of coverslip relative to the slide throughout the entire squashing and staining process, from hammering to final coverslip mounting. Scale bar = 10 μm. C. Well-spread, but under-flattened chromosomes, evident by chromosomes present in multiple focal planes, i.e., blurry and in-focus chromosomes visible simultaneously. Common fix: See Figure 3A legend regarding “blurriness”. Scale bar = 10 μm. D-E. Ideal Squashes. Characteristics include: flat chromosomes all visible in same focal plane, chromosomes are well spread from each other, with chromosome ends often visible for identification/cytological mapping; however chromosomes are not over-stretched and are roughly even in thickness/width throughout spread. Scale bar = 10 μm. F. Ideal squash and stain. Example includes lacO integration site at cytological site 96C, staining with antibodies against Nup98 (red, originally described in Capelson et al., 2010) and LacI (green, Rockland 600-401-B05 ), recognizing LacI-Sec13 fusion protein bound to lacO 96C, and Hoechst staining (blue). Scale bar = 10 μm.

    16. If the squash is of good quality, proceed by dipping the slide into liquid nitrogen while holding the slide with large blunt forceps (not fine dissection forceps).
      1. Wait until fizzling/boiling in liquid nitrogen stops, and then remove slide.
      2. Carefully flip off coverslip with razorblade, taking care to not slide razorblade too far into the area containing the chromosomes as they can be scraped off the slide.
    17. Immediately place the slide in a Coplin jar containing Wash Solution.
      1. Collect multiple slides at this step.
      2. If you are not able to collect all larvae on the same day because of differences in genotype emergence timing etc., slides can instead be kept in a Coplin jar in PBS without Tween at 4 °C overnight (this appears to be compatible with most antibodies).
    18. When finished dissecting and squashing all slides, incubate the slides in Blocking Solution for 30-60 min.
      1. This can be done by exchanging the Wash Solution for 50 ml of the Blocking Solution in the Coplin jar containing the slides, or by adding the Blocking Solution directly to the slides, in the same manner as the antibody cocktails, as described below (if trying to conserve BSA or if experimenting with different blocking conditions containing precious reagents etc.)
      2. Optimize the blocking time for different antibodies if necessary.
      3. Blocking overnight is not advised, and if storing slides overnight before antibody treatment is necessary, it is preferable to stop at the prior step in PBS, without Tween, as described above.

  3. Primary antibody treatment
    1. Collect Blocking Solution from the Coplin jar to make primary antibody cocktail for next step (or use fresh), and save 1 ml (or more if necessary) in 1.5 ml tube at 4 °C overnight for secondary antibody cocktail(s) for the following day.
    2. Make antibody cocktail(s), using 30 μl per slide (and making enough for one additional slide to account for pipetting inaccuracies), with primary antibodies generally starting at a 1/100 (volume/volume) dilution in Blocking Solution.
      1. Antibody dilutions for polytene chromosome spreads are generally 10x more concentrated relative to Western Blot dilutions.
      2. Extreme dilutions for particularly weak or strong antibodies can get up to 1/20 or down to 1/500, respectively.
      3. If there appears to be much nonspecific interband signal, try diluting the antibody.
      4. Antibody cocktail and other volumes would need to be adjusted if different sized coverslips are used.

        Notes:
        1. Fixation time in the standard fixation solution can vary between 30-120 s and may need to be optimized if 1 min fixation leads to antibody staining that is not adequate.
        2. For some antibodies, especially those against antigens that may be somewhat extracted from chromatin by this acetic acid-based fixation method (such as histone proteins, for instance), a different fixation strategy may be required in order to better preserve the target epitope, such as the following:
          1)
          Glands are first pre-fixed for 30 s in 2% PFA alone.
          2)
          Glands are then transferred to 2% PFA/45% Acetic Acid for 2 min.
          3)
          Squash/spread glands in a drop of 45% acetic acid as described previously.
          4)
          After freezing slide in liquid nitrogen and removing coverslip, place directly into cold 70% ethanol at -20 °C for a minimum of 30 min.
          For many antibodies, glands can be left overnight in ethanol and staining will be unaffected.
          5)
          Once all slides are collected and have been in ethanol at least 30 min, wash twice with PBST at room temperature to completely remove ethanol and proceed with staining normally.
          6)
          The timing of these steps, as well as solution strengths, may need to be optimized for various antibodies.

    3. Remove the slide from the Coplin jar, carefully remove excess PBST from the slide in the area outside the sharpie-outlined square by using a kimwipe or other tissue-style wiper, and pipette 30 μl of the antibody cocktail onto the outlined area.
    4. Carefully lower down an untreated coverslip onto the outlined area containing the antibody cocktail, using a pair of fine forceps, starting with coverslip at an angle at one edge of the outlined area and slowly lowering down/flattening the coverslip out, avoiding bubbles.
    5. Gently place slide on top of a slightly wet (but not soaking) paper towel in a closeable humidity chamber.
    6. Once all slides have been treated with primary antibodies, covered with coverslips, and placed in the humidity chamber, carefully close the chamber and gently place somewhere flat and undisturbed at 4 °C overnight.
      a. Drying out of the tissue should be avoided at all costs as it leads to high background and poor staining.
      b. Some antibodies can be incubated at room temperature for 3 h instead of overnight at 4 °C.

  4. Washing and secondary antibody treatment
    1. Carefully slide coverslips from slides using a gentle movement with a gloved finger in a direction parallel to the slide, and wash slides by putting them in a Coplin Jar containing Wash Solution.
      Note: If coverslips do not easily slide off with minimal friction and effort, the antibody cocktail may have dried out and that slide may not be useable, so take note and compare to other slides under same conditions when imaging to ensure quality.
    2. Wash the slides 10 min in Wash Solution, 3 times, either stationary on a bench top, or gently mixing on an orbital shaker at 100 rpm.
    3. Introduce fluorescently conjugated secondary antibodies (1/300 is a good working dilution, adjust as needed) in the same manner as with the primary antibodies, and incubate in a closed humidity chamber for 1h at room temperature in the dark (a bench drawer works well, just close and open slowly).

  5. Hoechst/DAPI treatment and coverslip mounting
    1. Carefully remove the coverslips and wash the slides again with Wash Solution 3 x 10min in the dark.
    2. Stain slides with Hoechst or DAPI (1/1,000 in PBS) for 2 min by adding 100 μl of the Hoechst/PBS cocktail onto the outlined area (do not cover with coverslip) and protecting slides from light, for example by covering with a tube rack cover lined with foil.
    3. Wash slides in PBS without Tween for 10 min in the dark.
    4. Remove excess liquid from around the outlined area of each slide with a wipe, and add 5-10 μl of Prolong Gold Anti-fade (or similar mounting medium) in one quadrant of outlined area.
    5. Carefully lower untreated coverslip onto the outlined area, and avoid bubbles by angling the coverslip so it contacts the quadrant with the mounting medium first, and then slowly lower to the rest of outlined area until flat.
    6. Carefully place nail polish on the coverslip corners to secure the slide, cover the coverslips, and wait ~5 min to allow the nail polish to dry.
      Note: Any forceful movement of the coverslip at this stage, or at any point after the chromosome fixation and flash freezing, will lead to chromosome stretching and tears, and should be avoided at all costs.
    7. When the nail polish at the coverslip corners is dry and the coverslip does not move, carefully seal the coverslip completely along the edges with the nail polish.
      Note: If time is not a concern, letting slide dry for 30 min will allow the mounting medium (if a hardening medium) to dry and make coverslips even more secure before completely sealing.
    8. Let the nail polish dry for a minimum of ~10 min (covered) and proceed to imaging, or store at 4 °C.
    9. Quality of slides will be best if imaged within one week (some antibodies will persist longer but the fluorescence signal of secondary antibodies will diminish over time).

  6. Imaging
    1. Image slides using a 60x objective lens for a large field of view, or a 100x objective lens with 1.6x magnification for the greatest zoom, using exposure levels or laser intensities low enough that no channels are overexposed, but such that brightest pixels are still bright.
    2. Image all slides under the same acquisition conditions, determined by setting the imaging settings on the brightest samples.
    3. Imaging on widefield microscopes is sufficient for most purposes, and will be less likely to photobleach the samples than using a laser scanning confocal will; however fine-scale chromatin structure, and also signal from some weaker antibodies, are better resolved using confocal imaging.
    4. Collect images of 10+ different sets of chromosomes per slide, and image 3 slides per condition/genotype.
    5. If saving files in proprietary formats such as .lif (Leica) or .czi (Zeiss), make sure to split the channels and save into tiff files prior to analysis in MATLAB by using a program such as ImageJ etc.
      Note: Otherwise, save tiff images as separate channels directly using image acquisition software before concluding image collection.

Data analysis

Here we present instructions for running the custom MATLAB program PCCcalc (see Supplement File 1) to analyze fluorescence intensity correlations. The program calculates the Pearson Correlation Coefficient (PCC) on a pixel-by-pixel basis between each pair of channels in three-channel images of chromosome squashes. In these examples, channels are Hoechst (or DAPI), a LacI-tethered protein at a single lacO integration site (the “tester” channel), and a “target” channel representing a protein or complex whose enrichment at the lacO integration site is being assessed (Figure 3F). The LacI-tethered protein forms a bright band on the polytene chromosome. PCCcalc uses the LacI signal to create a threshold image mask. The pixels defined by the mask are used to assess the correlation of fluorescence intensities. Files/images of the individual channels of the composite images used as an example are included as Supplement Files 2, 3, 4 [which are images of immunofluorescent staining with Hoechst (blue), Lac-I (green), and Brm (red, originally described in Nakayama et al., 2012)] for input into the PCCcalc function of MATLAB to follow along with the protocol referenced in this section.
  PCCcalc is designed to be executable by a novice. It can be used to measure intensity correlations in any three-channel data where a mask covering a region of interest can be defined by a single threshold intensity in the tester image. The current code includes the use of the DAPI/Hoechst channel to define the mask, in the event that the tester signal spreads off of chromatin/across multiple bands.


  1. Add the PCCcalc file to the MATLAB path so MATLAB can access the program, from here on referred to as ‘function’
    1. The MATLAB path can be determined by inputting “userpath” into the MATLAB command line.
    2. The name of the PCCcalc file must not differ from the function name (denoted by the word following the = sign within the first line of the function file), or MATLAB will not be able to find and run the function.
  2. Run the function by inputting into the MATLAB command line the function name, “pearsonCorr = PCCcalc” (without quotations).
  3. A dialog box will appear, to enable navigation to and selection of a folder containing the image data to be analyzed. The target folder must contain .tif(f) files of images containing individual fluorescence channels, with names containing separate channel designations, for example ch00, ch01, ch02.
  4. When prompted, enter the channel designations/”strings” corresponding to channel identifiers in the file names.
    e.g., ch00 for blue, ch01 for green (tester), ch02 for red.
  5. An RGB image will appear showing Hoechst in blue and the tester in green, with crosshairs. Click on the tester band/band to be analyzed.
  6. A BW (Black and White) image of the Hoechst (DNA) stain should appear. The displayed image is from a smaller, cropped form generated to focus on the band of interest, and only objects visible in this cropped image will be available for data analysis proceeding forward. (The size of this "plaquette" is hard-coded at 105 x 105 pixels but can be adjusted based on individual needs).
    1. Press ‘t’ to toggle between this image and a BW image of the tester/green channel.
    2. Look for the chromatin band in the Hoechst stain corresponding to the chromatin band containing the tester/green signal.
    3. When ready, press ‘s’ to bring up the Hoechst image and crosshairs.
    4. Click a spot along the edge of the target Hoechst band, and this will select a pixel whose intensity will be used to generate an initial Hoechst mask.
  7. A window, the Mask Selection Figure, with four images will appear (Figure 4). The upper images are BW images upon which are superimposed the outlines of the Hoechst (left) and green tester (right) masks. The lower left image shows the Hoechst mask (magenta), the tester mask (green), and the overlap between the two (white). The lower right image is an overlay of the images from the Hoechst (magenta) and tester (green) channels, with the overlapping Hoechst and tester masks outlined in cyan. The region within this cyan boundary is the final candidate region for calculating the correlation coefficient between the fluorescence signals therein. The inclusion of the Hoechst mask ensures that no signals, if any, present off of the DNA will be incorporated in the calculations.


    Figure 4. Mask Selection Figure. An example image from the MATLAB data analysis program “PCCcalc” (Supplement File 1) generated during the mask creation procedure used to designate the fluorescence area to analyze, with the mask defined by the band of interest (e.g., LacI fusion protein) and the Hoechst DNA stain for chromatin boundaries.

  8. The Hoechst and tester masks can be manually adjusted using the following commands from the keyboard:
    key command
    a   decrease current Hoechst mask by erosion
    s   increase current Hoechst mask by dilation
    d   decrease current Hoechst mask using higher threshold
    f    increase current Hoechst mask using lower threshold
    u   restart selection of Hoechst mask using candidate mask selected in Step 6d. above
    z   decrease current tester mask by erosion
    x   increase current tester mask by dilation
    c   decrease current tester mask using higher threshold
    v   increase current tester mask using lower threshold
    j    restart selection of tester mask using initial candidate mask
    k   restart process and return to Step 6d above
    g   draw a polygon by hand corresponding to the Hoechst region of interest
    i    skip this image
    r    save masks and proceed to calculate correlation coefficient

  9. If the manipulations of the Hoechst mask do not adequately limit the defined overlap to the band of interest present on DNA, press g to draw a Hoechst mask in manual mode. A new window will open with a BW image of the Hoechst channel.
    1. Use the crosshairs to draw a polygon around the DAPI band containing the tester band of interest, clicking at positions around the DAPI band corresponding to points of the desired polygon.
    2. Click back on the first point when finished. Individual points can be adjusted by dragging with the mouse, and the entire polygon can be moved by clicking within the shape and dragging.
    3. When finished, right-click inside the polygon and select “Create mask” from the popup menu. The window closes and the new Hoechst mask appears in the Mask Selection Figure (referred to in Step 7 above).
    4. Now use the following commands to compare and accept or reject the hand-drawn mask:
      key command
      t   toggle between the new hand-drawn mask and the previous automatically computed mask, or previously generated hand-drawn mask if multiple are attempted.
      p   redraw the mask.
      g   accept the hand-drawn mask, exit manual mode, and return to step H command options.
      l    reject the hand-drawn mask, use the previous mask and return to manual adjustments above.
  10. Once the masks are acceptable, press ‘r’.
    If masks cannot satisfactorily be made that accurately represent the band of interest, the current image can be skipped by pressing ‘i'.
  11. If 'r' has been pressed, then the Mask Selection Figure closes and a new figure with five panels, the Analysis Results Figure (Figure 5), opens.


    Figure 5. Analysis Results Figure. Found in MATLAB data analysis after mask creation is completed, this window displays the image being analyzed, the mask used for analysis, and graphs relating fluorescence intensity values of signals from two fluorescence channels at a time [Red-Green(RG), Green-Blue(GB), and Red-Blue(RB)], where each data point represents one pixel. The resulting Pearson Correlation Coefficients (“Corr Coeff”) derived from those relationships are displayed on the top of each graph. The immunofluorescence staining in the top left image includes Lac-I (green), Brm (red), and Hoechst (blue).

    1. The top row displays an RGB image (left) of all three channels (as defined in the first step, e.g., red = target, green = tester, blue = Hoechst) and the final overlap mask (right).
    2. The lower row shows three scatter plots with each dot representing an individual pixel within the mask, with its x and y values corresponding to the intensities of the respective fluorescence signals. The overall PCC for each pair of channels, calculated from the relationship between all the points in each plot, is then calculated, and displayed above each plot.
    3. The cropped images, the results figure, and the correlation coefficients are all automatically saved in a subfolder called “autocrop” generated in the directory folder.
  12. Press any key to continue to repeat with the next image/set of channels (starting with step 5).
  13. Repeat until all images in the folder have been analyzed, and then a table will show up the in MATLAB workspace called pearsonCorr, and will be automatically saved as a file in the “autocrop” subfolder (titled “pearsonCCs.mat”) with the correlation values for that set of images.
    1. Each column corresponds to the correlation values for one image, and each row a different pair of channels, in order of image analysis.
      row 1: red-green correlations
      row 2: green-blue correlations
      row 3: red-blue correlations
    2. These values should then be copied into a file in your preferred graphing/statistical analysis software (e.g., Excel, Prism etc.) for later processing before proceeding to another data set or completing analysis, as the generated pearsonCCs.mat file with the correlation values is MATLAB-specific and will not be accessible on a computer without MATLAB software and license.
    3. We found it is best to use a minimum of 10 images per slide, 3 slides (larvae) per genotype/condition.
    4. To compare the distributions of correlation coefficients between conditions/genotypes, perform a Fisher z-transformation of the correlation coefficients r using the following:

      z = 0.5 ln [(1 + r)/(1 - r)]

      The distributions of the transformed correlations z can then be compared using a t-test or one-way ANOVA. If using Prism, a one-way ANOVA test is a robust statistical analysis for these data with 3+ conditions/genotypes, using Tukey’s multiple comparisons post-test.

Recipes

  1. PBS (Cold Spring Harbor Labs Recipe)
    Reagent     Amount for 1x      Final 1x Concentration     Amount for 10x Stock       Final 10x Concentration
    NaCl                  8 g                         137 mM                            80 g                                    1.37 M
    KCl                    0.2 g                       2.7 mM                             2 g                                      27 mM
    Na2HPO4         1.44 g                     10 mM                             14.4 g                                100 mM
    KH2PO4            0.24 g                     1.8 mM                            2.4 g                                  18 mM
    Note: pH to 7.4 at 1x with HCl.
  2. PBST
    PBS + 0.1% Tween 20 diluted from 20% storage solution
  3. Fixation Solution: 2% PFA/45% Acetic Acid
    16% PFA diluted to 2% and 100% Acetic Acid diluted to 45% together in Milli-Q H2O
    **Fixation solution freshly made every 30 min of dissection
    ***16% PFA stored at 4 °C after opening the ampule and used within 3 weeks or chromosome structure preservation will be impaired
  4. 45% acetic acid
    100% Acetic acid diluted to 45% with Milli-Q H2O
    **Freshly made every 30 min of dissection
  5. Blocking solution/Antibody cocktail base
    PBS + 0.1% Tween 20 + 3% BSA (BSA dilution is mass per volume, so grams per ml)

Acknowledgments

This protocol was originally published in a research manuscript by Kuhn et al. (2019). We thank members of the Capelson and Little labs for their input on the research project that prompted development of this method, and J. Aleman for assistance in photo collection. M.C. is supported by the Research Scholar Grant RSG-15-159-01-CSM from the American Cancer Society and by NIH R01GM124143.

Competing interests

Authors declare no competing interests.

References

  1. Cai, W., Jin, Y., Girton, J., Johansen, J. and Johansen, K. M. (2010). Preparation of Drosophila polytene chromosome squashes for antibody labeling. J Vis Exp (36). DOI: 10.3791/1748.
  2. Kuhn, T. M., Pascual-Garcia, P., Gozalo, A., Little, S. C. and Capelson, M. (2019). Chromatin targeting of nuclear pore proteins induces chromatin decondensation. J Cell Biol 218(9): 2945-2961.
  3. Paro, R. (2008). Mapping protein distributions on polytene chromosomes by immunostaining. CSH protocols 2008: pdb.prot4714. 
  4. Capelson, M., Liang, Y., Schulte, R., Mair, W., Wagner, U. and Hetzer, M. W. (2010). Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell 140(3): 372-383.
  5. Nakayama, T., Shimojima, T. and Hirose, S. (2012). The PBAP remodeling complex is required for histone H3.3 replacement at chromatin boundaries and for boundary functions. Development 139(24): 4582-4590.

简介

[摘要] 果蝇几十年来,幼虫唾液腺多线染色体压片被用来分析全基因组蛋白质结合模式、转录激活过程以及特定基因位点染色质结构的变化。在过去的几年中,压榨方案已经有了许多改进,次优的重复性和较低的样本成功率是公认的警告。然而,当多线染色体用于更高通量的方法,如遗传或抗体筛查,或用于需要高质量染色体结构保存的实验时,低样本成功率是一个明显的缺点。在这里,我们提出了一个特别可重复的挤压和荧光染色协议,它可以在扩散良好的染色体上生成高质量的荧光图像。接下来是我们的新的、半自动的MATLAB分析程序,用于以逐像素的方式确定多线染色体上单个位点上感兴趣的荧光信号之间的相关性。在我们的案例中,我们已经用这种方法来评估染色体在基因组位点的变化,这些位点被核孔蛋白的异位靶向。我们的分析程序的使用提高了对染色质结构的变化或染色质的蛋白质补充的无偏结论的能力,而不考虑免疫荧光染色的样本变化。由于它只是基于特定位置的荧光强度的差异,因此所提供的分析程序不局限于对多线染色体的分析,并且可以应用于许多不同的上下文中,其中任何特定位置的荧光信号之间的相关性是感兴趣的。

[] 来自果蝇幼虫唾液腺的多线染色体具有异常大的尺寸(在典型的细胞培养显微镜下可见)。这是由于细胞周期过程的改变,每一条染色体都有大约1000个拷贝,而这些拷贝并不彼此分离。由此产生的大染色体有定型的和可重复的交替带的浓缩和去浓缩染色质带,因为染色体排列整齐,并且在多化处理时可以看到特定位点的染色质结构。这些染色体的性质使得对染色质结构的分析相对于二倍体细胞类型是唯一可见的,能够分析整个发育、转录激活或扰动时的染色质变化。此外,通过多线染色体的免疫荧光染色,可以观察蛋白质的全基因组和位点特异性染色质结合(Paro,2008;Cai等人,2010),从而可以研究候选蛋白质与染色质凝聚、转录活性和组蛋白修饰之间的关系。以这种方式,多线染色体染色提供了一个快速和廉价的替代品或分子分析的补充,如染色质免疫沉淀,结合测序(ChIP-seq)。

我们的压片和荧光分析管道专门设计用于分析单个感兴趣条带的染色质结构和蛋白质招募/变化,最初是针对与基因组lacO重复序列结合的LacI栓系蛋白质带(Kuhn等人,2019年)。在这项研究中,我们针对特定的核孔组分,融合到LacI和lacO阵列上,整合在基因组中的特定位置。然后我们的目标是确定这种靶向性是否会导致候选相互作用伙伴的招募,以及靶染色质致密性的变化。这种方法涉及到在整个扩散染色体基因组中识别单个感兴趣的带(包含lacO位点),这需要高质量的染色体扩散,以提高染色体带型的可见性和分析。此外,通过多个抗体进行筛选,以检测许多相互作用的候选伙伴,需要一个极为重复的压片方案,以最小的样本失败率。为了实现这些目标,我们开发了优化的多线压缩协议,该协议可用于提高图像质量和样本成功率。它还特别适用于染色体的“可映射性”(通过对多线带模式的细胞学定位来确定基因组位置)和更高吞吐量的应用。

为了可靠地分析染色质结构的位点特异性变化(即通过DNA染色荧光强度评估的染色质凝聚变化)和蛋白质补充,我们开发了一个半自动的MATLAB函数。这个函数,PCCcalc,以一个像素为基础,以无偏的方式计算在一个确定的基因组位点上的多个荧光信号之间的Pearson相关系数(PCC)。这大大减少了数据分析的时间和由手动分析和基于用户的可变性而产生的主观性。此外,这种方法避免了荧光背景或图像采集中逐段变化的影响,因为分析的重点是荧光信号之间的关系,而不是给定信号的绝对值。该方法可用于多线染色体上任何易于识别的位点,以确定多个蛋白质因子之间的结合相关性,或找出感兴趣的蛋白质与染色质致密性相关变化之间的相关性。此外,我们相信这种分析方法可以扩展到测定荧光相关性,而不仅仅局限于在多线染色体上的应用,而且可以用于分析荧光标记生物标记在许多其他环境和细胞类型中的共定位或共现。

关键字:多线染色体按压, 染色质结构, 蛋白募集, Pearson 相关系数, 免疫荧光分析

材料和试剂


 


1.     滤纸(Fisherbrand,目录号:05-714-4)


2.     带橡胶头的锤子(Graham Field,目录号:1312-1)


3.     好夏比


4.     1.5毫升试管(美国科学,目录号:1615-5510)


5.     解剖井盘/Pyrex斑点板(Fisher Scientific,目录号:13-748B)


6.     Dumont Fine#55镊子(精细工具,目录号:11295-51)


7.     盖玻片(Fisherbrand,目录号:12-542A)


8.     科普林罐(VWR,目录号:74830-150)


9.     湿度箱(如Pyrex盖玻璃烤盘)


10.  聚赖氨酸处理的载玻片(Polysciences,目录号:22247)


11.  聚赖氨酸(上述预处理载玻片的替代品)(Sigma-Aldrich,目录号:P8920-100 ml)


12.  西格玛科特(Sigma-Aldrich,目录号:SL2-100ML)


13.  磷酸盐缓冲盐水(PBS)试剂:


a、 NaCl(Fisher,目录号:BP358-212)


b、 KCl(费希尔,目录号:BP366-500)


c、 Na2HPO4(西格玛,目录号:S3264-500G)


d、 KH2PO4(西格玛,目录号:P9791-1KG)


14.  使用的主要抗体:抗LacI(Rockland,目录号:600-401-B05)、抗Nup98(Capelson等人,2010年)、抗Brm(Nakayama等人,2012年)


15.  吐温20(费希尔,目录号:BP337-500)


16.  冰醋酸(Fisher,目录号:BP2401-212)


17.  16%多聚甲醛(PFA)(Alfa Aesar,目录号:43368)


18.  液氮


19.  Kimwipes或其他纸巾式雨刷器(Fisher Scientific,产品目录号:06-666A)


20.  荧光二级抗体(如Thermo Fisher,目录号:A-11004)


21.  Hoechst 33342(赛默飞世尔,目录号:H3570)(10毫克/毫升储备)


22.  延长金防褪色(赛默飞世尔,产品编号:P36930)


23.  PBS(见配方)


24.  PBST(见配方)


25.  固色液(见配方)


26.  45%醋酸(见配方)


27.  阻断液/抗体鸡尾酒碱(见配方)


 


设备


 


1.     镊子


2.     微量加样器


3.     化学气罩


4.     解剖显微镜(如徕卡,型号:徕卡S6E)


5.     细胞培养/相差显微镜(如徕卡,型号:徕卡DM IL)


6.     宽场荧光显微镜(如徕卡,型号:徕卡DM6000)和/或激光扫描共焦显微镜


7.     (可选)轨道振动筛(VWR,目录编号:12620-938)


软件


 


1.     MATLAB(许可证)(分析必需)


2.     绘图/统计分析软件,如Excel、Prism(许可证)(分析所需)


三。(可选)ImageJ(免费)三。(可选)ImageJ(免费)


 


程序


 


A、 预先制备Sigmacote处理过的盖玻片和聚赖氨酸处理的载玻片


为了防止染色体贴壁和多聚赖氨酸染色玻片的附着,应注意:为了防止染色体的粘连,应优先使用聚赖氨酸处理过的玻片。最好在实验前处理。


 


Sigmacote处理的盖玻片


1.     将一个1.5毫升的管架(或类似结构)用塑料包装起来,放在一个更大的矩形纸巾上,放在一个化学品罩中,见图1。


2.     将Sigmacote(储存在4°C)倒入Sigmacote瓶盖。


3.     使用干净、精细的镊子,将盖滑片单独浸入Sigmacote中,完全浸没。最好使用弯曲/弯曲的尖端,这样镊子就可以最小限度地接触到盖玻片(参见下面的步骤B7)。


4.     立即取下盖玻片,将盖玻片的一角接触到Sigmacote瓶盖中的液体表面,以除去多余的液体,并将盖玻片以约60°的角度放置在纸巾顶部的塑料覆盖架上,以使任何剩余的Sigmacote灯芯远离盖玻片(见图1)。


5.     完成所需数量的盖玻片后,将Sigmacote从瓶盖中放回Sigmacote瓶中,在4°C下储存,在1年内使用。


6.     将盖玻片放在化学品罩中晾干一夜(如果时间有限,可在约1小时后使用)。


7.     将盖玻片存放在4°C的环境中(隔夜干燥后再放回原容器即可),最好在2个月内使用。


 


 


图1。一个简单的实验室装置,用于干燥Sigmacote处理后的盖玻片。如上文A1-A4所述,将用于多线染色体压片的盖玻片用Sigmacote处理,并靠在塑料包装的1.5毫升管架上,放在纸巾上,放在化学品罩内。


 


聚赖氨酸处理的载玻片


可购买经预处理的聚赖氨酸载玻片(我们发现,与自处理的载玻片相比,经预处理的载玻片具有更低的自荧光背景),但为了节省成本,或者如果无法获得预处理的载玻片,则可使用聚L-赖氨酸处理标准磨砂显微镜载玻片,其处理方式与使用Sigmocote处理的盖玻片类似:


1.     将聚L-赖氨酸倒入一个玻璃Coplin罐中。


2.     插入磨砂显微镜载玻片,使载玻片的非磨砂玻璃部分(即,不包括磨砂标签区域)浸没在聚赖氨酸中。


3.     一旦Coplin罐装满载玻片,按处理时的原始顺序取出载玻片,并与纸巾上的垂直面(如锥形架)成~60°角。


4.     让载玻片干燥一夜,如有需要,至少2小时。


5.     储存在4°C下(隔夜干燥后可放回原滑箱中),最好在2个月内使用。


 


B、 腺体切割和染色体压扁


在开始解剖之前,收集所有材料,制作固定溶液,并在适当的容器中填充液氮。


1.     挑选理想的3龄游荡幼虫,并将最多4只放入PBS中的玻璃分离井盘中。


 


注:挑选幼虫是获得最高质量南瓜最重要的一步!用于收集唾液腺的3龄游荡幼虫应为:


a。不拥挤的瓶子/瓶子–理想的幼虫对营养素的竞争很低,而且又肥又健康。


b。晚游荡3龄,最小移动,但还没有在前蛹阶段,因为较老的幼虫(主要只移动嘴)将有更大的染色体和最好的南瓜。


(一)仍在瓶/瓶壁上快速移动的幼虫将更年轻,多线染色体复制较少,因此染色体更小。


(二)然而,太接近化蛹期/蛹前期的幼虫会产生过多的唾液腺“胶”蛋白,这会导致扩散困难和免疫荧光背景增强。为了避免这种情况,大的“玻璃状”或更透明的唾液腺(其中充满了刺穿时释放的胶状物质),(见图2)应该跳过,以获得最佳质量的南瓜。不要吃前呼吸孔延长的幼虫,因为它们几乎总是有这种类型的腺体。


c。从游荡幼虫的第一波开始,通常在小瓶/杂交体建立或翻转后6-7天出现(如果在21-22日饲养摄氏度室温)。


优质南瓜可以来自在无拥挤条件下游荡第二天的幼虫,但是唾液腺和染色体通常是第一波幼虫最大的,所以成功率/质量将从第一波幼虫中最高。


d。女性(除非对性别差异感兴趣)。雌性幼虫的唾液腺和多线染色体往往比雄性幼虫大。3龄幼虫的雌性性别是由幼虫内部两侧(距幼虫后端约1/3)看不到大而透明的球形性腺而决定的。这些大的性腺代表男性。


 


 


图2。从3龄游荡幼虫中分离出不同类型的唾液腺。 A、 典型的一组唾液腺,外观通常不透明/乳白色,可见清晰的细胞。B、 成熟腺体应避免,取自接近化蛹期的幼虫,由于粘着物质增加,通常细胞边界不太明显,因此显得更加清晰/透明/“玻璃状”。附着在腺体上的白色碎片是剩余的脂肪体。


 


2.     将一只幼虫转移到带PBS的新井中进行解剖。每2-3次解剖后,更换该井的PBS。


3.     用一把钳子抓住幼虫的口钩,用另一把钳子从身体的一半左右轻轻地向另一个方向拉动幼虫,以此来解剖唾液腺。(为了演示果蝇幼虫唾液腺的解剖,这里有很多公开视频)


4.     从腺体侧面剥下脂肪体(白色组织条),因为这有助于下游成像中的荧光背景;但是,如果这些脂肪体持续存在,则不要花费超过几分钟的时间移除这些脂肪体,最好尽快进行固定。在固定前解剖不应超过5分钟。


5.     将腺体转移到另一个含有~100-200μl新制固定液的分离皿中,静置1分钟。


a、 腺体可以由“叉形骨”或茎干区固定,因为这样可以防止损坏腺体细胞。


b、 使用一对带有弯曲/弯曲尖端的镊子(如下面的步骤B7所述)可以更容易地将腺体转移到这里。


c、 醋酸在这里起到破坏核膜和帮助染色体扩散的作用,但是并不是所有抗体在这些条件下都能很好地工作,有些抗体可能需要在不同的固定长度或溶液中进行优化,见c节中的注释。


注意s:


a。在我们手中,所有的醋酸溶液必须每30-45分钟用100%的醋酸制成新鲜溶液,以确保染色体的正确传播,因为醋酸稀释液很快变质,会使染色体更难传播。


b。另外,一旦打开,16%的PFA必须在4℃下保存(通常从玻璃安瓿转移到15ml锥形管中),并且仅使用3周,以获得最佳染色体扩散和结构保存。


6.     重要!从这一点开始,迅速而小心地进行固定,直到玻片在液氮中冻结(下图)。在上述固定培养期间,最好准备好盖玻片并贴上标签。


7.     用kimwipe或其他纸巾擦拭Sigmocote处理过的盖玻片上的残留物,在盖玻片上加入7μl新鲜的45%乙酸,然后轻轻地将脆弱的腺体转移到乙酸滴中。


a、 腺体在固定后变得异常脆弱,需要巧妙地转移。


b、 因为这个步骤是一对镊子,一个弯曲的尖端,这样它在面向另一个尖端的末端形成一个弯曲或钩状的形状,防止镊子完全闭合。它们可以在顶端的一小滴液体中拾起腺体,防止腺体被挤压和粘在镊子上。这些可以用于所有的转移步骤,但在固定后当腺体变得脆弱时尤其必要。强烈推荐


8.     将经聚赖氨酸处理的载玻片轻轻放在乙酸滴上,使腺体大约在载玻片的中间。


9.     翻转载玻片,用戴手套的食指在盖玻片上施加压力,同时将盖玻片“推入”载玻片,同时将盖玻片以顺时针方向旋转约1厘米的圆圈,绕3圈,分裂组织,扩散细胞和染色体(步骤B9-B14见视频1)。


a、 重要的是始终在一个方向上绕一个圈,以防止染色体缠结。


b、 盖玻片滑动应相对平稳,不会卡住。如果发生卡滞,请参阅以下注释。


c、 这一步需要优化和练习,让每个人学会适当的动作和手指的压力,以获得最佳的伸展。图3A显示了当这个步骤需要更多压力时染色体的样子。




 


注:移动盖玻片的目的是充分分离细胞,使它们不重叠,使细胞膜破裂,并使染色体扩散,但在足够小的区域内,所有染色体仍能被盖玻片覆盖,以便以后安装和成像。如果盖玻片在扩散过程中滑动不顺畅,这将导致染色体撕裂/拉伸(图3B)。然而,阻力太小,细胞就不会断裂,染色体也不会充分扩散(图3A),这可能是由于盖玻片和玻片之间的液体过多造成的。


有助于防止封面纸粘住的措施:


a。避免使用透明的“胶状”压盖(如上步骤B1中所述,这些压盖呈玻璃状/透明状)(见图2)。


b。使用新制的、完全干燥(最好隔夜)并完全擦掉Sigmacote处理过的盖玻片,使用新鲜的Sigmacote制成(如果您自己处理,也适用于聚赖氨酸处理的玻片)。


c。使用30-45分钟内制备的新鲜醋酸溶液。


 


 


视频1。多线染色体南瓜


 


10.  翻转载玻片,使盖玻片在底部,然后小心地将玻片插入一张折叠的厚滤纸中。


11.  用两个手指握住滑动盖所在位置的两侧,以固定滑块并防止滑块和滑动盖之间发生横向移动。


12.  用橡胶锤以中等力度敲击盖滑动区域约30次。这一步需要练习,所用的力需要针对每个人进行优化。


13.  直接向下推载玻片,将两个拇指牢牢地放在盖玻片的两侧,不要让盖玻片相对于载玻片滑动(见视频1),以便在成像时使染色体变平并防止3D/失焦染色体结构(图3A和3C)。


14.  从滤纸上取下载玻片,并在玻片背面用精细的锐利标记盖玻片角落的位置,以便在将来的抗体培养和安装中定位染色体区域。


15.  在细胞培养/相差显微镜下,在20倍或40倍物镜下检查染色体的传播质量。如果展开令人满意(图3D-3E),则继续。否则,将载玻片扔掉,对下一个幼虫重复,确保每2-3个幼虫都能很好地改变解剖中的PBS。


 


注:染色体扩散的质量控制:


a。一个好的染色体传播应该有ch呈扇形散开、有点分离、完好无损、未拉伸/撕裂且平坦(图3D-E)。


b。如果幻灯片显示大部分染色体撕裂/拉伸(图3B),染色体非常缠结在一起(图A-B),或细胞核没有充分断裂,染色体仍在一个紧密的球体中(图3A),通常很难从这些数据中获得有用的数据,并且继续进行不合标准的幻灯片通常是浪费时间和资源。


c。有了这个方案,一旦有人实践,我们通常有一个更高的成功率比以前尝试的方案,高达80-90%的经验,从理想的幼虫开始。但是,在这一阶段,您仍然可以扔掉几张幻灯片。由于压片和染色的固有变异性,您希望每种情况(基因型/抗体等)至少有3张高质量的玻片。


 


 


图3。染色体质量差的南瓜。 A、 扩散不足的南瓜,明显表现为缺乏从原始圆形核形状向外扩散的染色体;扁平化程度较低的南瓜,明显表现为存在于多个焦平面中的染色体,即同时可见模糊和聚焦的染色体。常见方法:为了增加染色体的扩散,在步骤B9中的盖玻片移动过程中必须使用更大的压力;为了减少“模糊度”,请确保在步骤B13中以更大的压力将盖玻片推入载玻片(避免盖玻片相对于载玻片的侧向移动)。比例尺=10μm。染色体被Hoechst染色(显示为白色),在A-E.B.过度伸展的染色体上,可见染色质带清晰的细链。染色质也会在某些地方分裂,没有带出现在他们的定型,有组织的秩序。常见解决方法:在步骤B12中,用锤子轻敲滑块,同时确保在整个挤压和染色过程中,从锤击到最终的盖滑安装,都要避免盖卡瓦相对于滑块的任何横向移动。比例尺=10μm.C.分布良好,但在扁平染色体下方,可见于多个焦平面中的染色体,即同时可见模糊和聚焦的染色体。常见修复:参见图3A“模糊”图例。比例尺=10μm。D-E。理想压片。其特征包括:在同一焦平面上都可见扁平染色体,染色体彼此间的扩散很好,染色体末端常可见,便于识别/细胞学作图;但染色体不会过度伸展,在整个扩散过程中,其厚度/宽度大致均匀。比例尺=10μm.F。理想的挤压和染色。与LacchSO结合位点的蓝染色(p96c)和LachsO-96位点结合的蓝染色(p96c)和LachsO-2010中的蓝染色(p96c)。比例尺=10μm。


 


16.  如果南瓜的质量很好,继续将载玻片浸入液氮中,同时用大钝镊子(不是细解剖钳)固定载玻片。


a、 等到液氮中的灰化/沸腾停止,然后取下滑块。


b、 小心地用剃须刀将盖玻片掀开,注意不要将剃须刀滑入含有染色体的区域太远,因为它们可能会从玻片上刮下。


17.  立即将载玻片放入装有洗涤液的Coplin罐中。


a、 在此步骤中收集多张幻灯片。


b、 如果由于基因型出现时间的不同而不能在同一天采集所有幼虫,则可以将载玻片放在PBS中的Coplin罐中,在4°C下放置过夜(这似乎与大多数抗体兼容)。


18.  所有载玻片解剖压碎后,在封闭液中孵育30-60分钟。


a、 这可以通过在装有载玻片的Coplin罐中用洗涤液换成50毫升的封闭溶液,或以与抗体鸡尾酒相同的方式将封闭溶液直接添加到载玻片中,如下文所述(如果试图保存牛血清白蛋白,或在含有贵重试剂的不同封闭条件下进行试验等)


b、 如有必要,优化不同抗体的阻断时间。


c、 不建议隔夜封闭,如果需要在抗体治疗前过夜储存载玻片,则最好在PBS的前一步停止,不使用吐温,如上所述。


 


C、 初级抗体治疗


1.     从Coplin罐中收集阻断液,制成下一步的一级抗体鸡尾酒(或新鲜使用),并在1.5 ml试管中4℃保存1ml(或更多,如有必要)作为第二天的二级抗体鸡尾酒。


2.     制备抗体鸡尾酒,每个玻片使用30μl(并制作足够多的一个玻片以说明移液不准确),一级抗体通常从阻断溶液中的1/100(体积/体积)稀释开始。


a、 多线染色体扩散的抗体稀释液通常比免疫印迹稀释液浓度高10倍。


b、 特别弱或强抗体的极端稀释可分别达到1/20或1/500。


c、 如果有许多非特异性带间信号,尝试稀释抗体。


d、 如果使用不同大小的盖玻片,则需要调整抗体鸡尾酒和其他体积。


 


笔记:


一。标准固定溶液中的固定时间在30-120秒之间变化,如果固定1分钟导致抗体染色不充分,则可能需要优化。


二。对于某些抗体,尤其是针对通过这种基于乙酸的固定方法从染色质中提取的抗原的抗体(例如组蛋白),可能需要不同的固定策略以更好地保存目标表位,例如:


(一)腺体首先在2%PFA中预先固定30秒。


(二)然后将腺体转移到2%PFA/45%乙酸中2分钟。


(三)如前所述,在一滴45%的醋酸中挤压/扩散腺体。


(四)在液氮中冷冻载玻片并取下盖玻片后,将其直接放入-20°C的70%冷乙醇中至少30分钟。


对于许多抗体,腺体可以在乙醇中过夜,染色不会受到影响。


(五)收集所有载玻片并在乙醇中放置至少30分钟后,在室温下用PBST清洗两次,以完全去除乙醇并继续正常染色。


(六)这些步骤的时间,以及溶液强度,可能需要针对各种抗体进行优化。


 


3.     从Coplin罐中取出载玻片,使用kimwipe或其他纸巾式擦拭器小心地从sharpie轮廓方格外的载玻片上去除多余的PBST,并用吸管将30μl抗体鸡尾酒移到轮廓区域。


4.     使用一对细镊子小心地将未经处理的盖玻片降到含有抗体混合物的轮廓区域,从覆盖片开始,在轮廓区域的一个边缘处形成一个角度,然后慢慢降低/压平盖玻片,避免产生气泡。


5.     将玻片轻轻地放在稍湿(但不是浸湿)的纸巾上,放在密闭的湿度室中。


6.     一旦所有载玻片都用一级抗体处理过,用盖玻片盖住,并放置在湿度室中,小心地关闭密封室,轻轻地将其放置在4°C的平坦、不受干扰的地方过夜。


a、 应不惜一切代价避免组织干燥,因为它会导致高背景和不良染色。


b、 有些抗体可以在室温下孵育3小时,而不是在4℃过夜。


 


D、 洗涤及二次抗体治疗


1.     用戴手套的手指沿与载玻片平行的方向轻轻移动,小心地从载玻片上滑出玻片,并将玻片放入装有洗涤液的科普林罐中清洗。


注:如果盖玻片不容易滑动,摩擦力和作用力最小,抗体混合物可能已经干透,该载玻片可能不可用,所以成像时请注意并与其他玻片在相同条件下进行比较,以确保质量。


2.     在洗涤液中清洗载玻片10分钟,3次,要么静止在工作台上,要么以100转/分的速度在轨道摇床上轻轻混合。


3.     以与初级抗体相同的方式引入荧光结合的二级抗体(1/300是一个很好的工作稀释液,根据需要进行调整),在封闭的湿度室中在室温下黑暗中培养1h(一个抽屉可以很好地工作,只需缓慢地关闭和打开)。


 


E、 Hoechst/DAPI处理和盖板安装


1.     小心地取下盖玻片,并在黑暗中用洗涤液3 x 10分钟再次清洗载玻片。


2.     用Hoechst或DAPI(PBS中的1/1000)对载玻片进行染色2分钟,方法是在轮廓区域添加100L的Hoechst/PBS鸡尾酒(不要用盖玻片盖住),并保护载玻片不受光照,例如用内衬箔纸的管架盖覆盖。μ


3.     将载玻片放入PBS中,在黑暗中洗涤10分钟。


4.     用抹布从每个载玻片的轮廓区域去除多余的液体,并在轮廓区域的一个象限中添加5-10μl长效金防褪色剂(或类似的安装介质)。


5.     小心地将未经处理的盖玻片放在轮廓区域,并通过倾斜盖玻片使其首先与安装介质接触象限,然后慢慢降低到轮廓区域的其余部分,直到其变平,从而避免产生气泡。


6.     小心地将指甲油放在盖玻片的角上以固定滑片,盖住盖玻片,并等待~5分钟使指甲油变干。


注意:在这一阶段,或在染色体固定和快速冷冻后的任何时候,盖片的任何用力移动都会导致染色体拉伸和撕裂,应不惜一切代价避免.


7.     当盖玻片拐角处的指甲油干燥且不移动时,小心地用指甲油沿边缘将盖玻片完全密封。


注意:如果时间不是问题,让滑块干燥30分钟将允许安装介质(如果是硬化介质)干燥,并使盖滑片在完全密封之前更加牢固。


8.     让指甲油干燥至少10分钟(覆盖),然后继续成像,或在4°C下储存。


9.     如果在一周内成像,载玻片的质量最好(有些抗体会持续更长时间,但次级抗体的荧光信号会随着时间的推移而减弱)。


 


F、 成像


1.     图像幻灯片使用60倍物镜获得大视场,或使用100倍物镜(1.6倍放大倍率)实现最大变焦,使用的曝光水平或激光强度足够低,没有通道被过度曝光,但最亮的像素仍然明亮。


2.     在相同的采集条件下对所有幻灯片进行成像,这是通过在最亮的样本上设置成像设置来确定的。


3.     在宽视场显微镜上成像对大多数目的来说是足够的,并且比使用激光扫描共焦显微镜更不容易使样品发生光漂白;然而,精细的染色质结构以及来自一些较弱抗体的信号,使用共焦成像能更好地分辨。


4.     每张幻灯片收集10+组不同染色体的图像,每个条件/基因型收集3张幻灯片。


5.     如果以.lif(Leica)或.czi(蔡司)等专有格式保存文件,请确保在使用ImageJ等程序在MATLAB中进行分析之前分割通道并保存为tiff文件。


注:否则,在结束图像采集之前,请直接使用图像采集软件将tiff图像保存为单独的通道。


 


数据分析


 


这里我们介绍了运行定制的MATLAB程序PCCcalc(see)来分析荧光强度相关性的说明。该程序以像素为基础计算染色体压片的三个通道图像中每对通道之间的皮尔逊相关系数(PCC)。在这些例子中,通道是Hoechst(或DAPI),单个lacO整合位点处的LacI栓系蛋白质(“测试员”通道)和代表蛋白质或复合物的“目标”通道,其在lacO整合位点的富集正在被评估(图3F)。LacI栓系蛋白在多线染色体上形成一条明亮的带。PCCcalc使用LacI信号创建一个阈值图像掩码。由掩模定义的像素用于评估荧光强度的相关性。作为示例使用的复合图像的各个通道的文件/图像包括为,[它们是带有Hoechst(蓝色)、Lac-I(绿色)和Brm(红色,最初在中山等人中描述)的免疫荧光染色的图像。,2012年)输入到MATLAB的PCCcalc函数中,以遵循本节中引用的协议。补充文件1补充文件234


PCCcalc被设计成可以由新手执行。它可用于测量任何三通道数据中的强度相关性,其中覆盖感兴趣区域的掩模可以由测试器图像中的单个阈值强度定义。当前代码包括使用DAPI/Hoechst通道来定义掩模,以防检测仪信号从染色质中散开/跨越多个频带。


 


1.     将PCCcalc文件添加到MATLAB路径中,这样MATLAB就可以访问程序,从这里开始称为“函数”


a、 MATLAB路径可以通过在MATLAB命令行中输入“userpath”来确定。


b、 PCCcalc文件的名称不能与函数名(在函数文件的第一行中用“=”号后面的单词表示)不同,否则MATLAB将无法找到并运行该函数。


2.     通过在MATLAB命令行中输入函数名“pearsonCorr=PCCcalc”(不带引号)来运行函数。


3.     将出现一个对话框,允许导航到包含要分析的图像数据的文件夹并对其进行选择。目标文件夹必须包含包含单个荧光通道的图像的.tif(f)文件,名称包含单独的通道名称,例如ch00、ch01、ch02。


4.     出现提示时,在文件名中输入对应于频道标识符的频道名称/“字符串”。


例如,ch00代表蓝色,ch01代表绿色(测试仪),ch02代表红色。


5.     一个RGB图像将出现,显示Hoechst为蓝色,测试仪为绿色,带有十字准线。单击要分析的检测仪波段。


6.     应该出现Hoechst(DNA)染色的BW(黑白)图像。显示的图像来自一个更小的裁剪表单,生成的目的是聚焦于感兴趣的区域,并且只有在这个裁剪的图像中可见的对象才能用于继续进行数据分析。(此“plaquette”的大小硬编码为105 x 105像素,但可以根据个人需要进行调整)。


a、 按“t”在该图像和检测仪/绿色通道的BW图像之间切换。


b、 在Hoechst染色中寻找与包含检测仪/绿色信号的染色质带相对应的染色质带。


c、 准备好后,按“s”键调出赫斯特图像和十字准线。


d、 点击目标Hoechst波段边缘的一个点,这将选择一个像素,其强度将用于生成初始Hoechst掩模。


7.     一个窗口,遮罩选择图,将出现四个图像(图4)。上面的图像是BW图像,上面叠加了Hoechst(左)和green tester(右)掩模的轮廓。左下角的图像显示了Hoechst掩模(洋红色)、测试器掩模(绿色)以及两者之间的重叠(白色)。右下角的图像是Hoechst(洋红色)和tester(绿色)通道的图像叠加,重叠的Hoechst和tester掩码用青色勾勒。该青色边界内的区域是用于计算其中的荧光信号之间的相关系数的最终候选区域。加入Hoechst掩模可以确保DNA中没有信号(如果有的话)也不会被纳入计算中。


 


 


图4。遮罩选择图。MATLAB数据分析程序“PCCcalc”()在掩模创建过程中生成的示例图像,用于指定要分析的荧光区域,掩模由感兴趣的带(如LacI融合蛋白)和染色质边界的Hoechst DNA染色确定。补充文件1


 


8.     可以使用键盘上的以下命令手动调整Hoechst和tester掩码:


按键命令


腐蚀降低电流的Hoechst掩模 


通过扩张来增加电流的Hoechst掩模 


d使用更高的阈值降低电流Hoechst掩模 


f使用较低阈值增加电流Hoechst掩模


u使用上述步骤6d.中选择的候选掩模重新选择Hoechst掩模


z腐蚀降低电流测试仪面罩


x放大电流测试仪面罩


使用更高的电流屏蔽测试仪降低电流阈值


使用低阈值增加电流测试仪掩模


j使用初始候选掩模重新选择测试仪掩模


k重新启动流程并返回到上面的步骤6d


用手画一个与Hoechst感兴趣区域相对应的多边形


我跳过这个图像


r保存掩码并继续计算相关系数


 


9.     如果Hoechst掩模的操作没有充分限制DNA上存在的感兴趣带的重叠,则按g以手动模式绘制Hoechst掩模。一个新的窗口将打开,显示Hoechst频道的BW图像。


a、 使用十字准线在包含感兴趣的测试仪带的DAPI带周围绘制一个多边形,单击DAPI带周围对应于所需多边形点的位置。


b、 完成后,单击“返回”第一个点。可以通过使用鼠标拖动来调整各个点,也可以通过在形状内单击并拖动来移动整个多边形。


c、 完成后,右键单击多边形内部并从弹出菜单中选择“创建遮罩”。窗口关闭,新的Hoechst掩模出现在掩模选择图中(参见上文第7步)。


d、 现在使用以下命令比较并接受或拒绝手绘遮罩:


按键命令


t在新的手绘遮罩和上一个自动计算的遮罩之间切换,如果尝试多次使用,则在先前生成的手绘遮罩之间切换。    


p重绘面具。


g接受手绘遮罩,退出手动模式,返回步骤H命令选项。


lR弹出手绘遮罩,使用上一个遮罩并返回到上面的手动调整。    


10.  一旦口罩合格,按“r”。


如果无法令人满意地制作出准确表示感兴趣波段的遮罩,则可通过按“i”跳过当前图像。


11.  如果按下了“r”,则遮罩选择图关闭,并打开一个包含五个面板的新图形,即分析结果图(图5)打开。


 


图5。分析结果图。在完成掩模创建后的MATLAB数据分析中,该窗口显示正在分析的图像、用于分析的掩模以及一次来自两个荧光通道的信号的荧光强度值的图形[红绿(RG)、绿蓝(GB)和红蓝(RB)],其中每个数据点代表一个像素。从这些关系得到的皮尔逊相关系数(“Corr Coeff”)显示在每个图表的顶部。左上角的免疫荧光染色包括Lac-I(绿色)、Brm(红色)和Hoechst(蓝色)。


 


a、 顶行显示所有三个通道(如第一步中定义的,如红色=目标,绿色=测试仪,蓝色=赫斯特)和最终重叠掩模(右侧)的RGB图像(左侧)。


b、 下一行显示三个散点图,每个点代表掩模内的单个像素,其x和y值对应于各自荧光信号的强度。然后计算每对地震道的总PCC(根据每个曲线图中所有点之间的关系计算得出),并显示在每个曲线图上方。


c、 裁剪后的图像、结果图和相关系数都自动保存在目录文件夹中生成的名为“autocrop”的子文件夹中。


12.  按任意键继续重复下一个图像/频道集(从步骤5开始)。


13.  重复此操作,直到文件夹中的所有图像都已分析完毕,然后一个名为pearsonCorr的表格将显示在MATLAB工作区中,并将自动保存为“autocrop”子文件夹(标题为皮尔逊公司.mat)的值。


a、 按照图像分析的顺序,每列对应一个图像的相关值,每行对应一对不同的通道。


第1行:红绿相关性


第2行:绿蓝相关性


第3行:红蓝相关性


b、 然后,应将这些值复制到您首选的绘图/统计分析软件(如Excel、Prism等)中的一个文件中,以便在进行其他数据集或完成分析之前进行后续处理,作为生成的皮尔逊公司.mat具有相关值的文件是MATLAB特有的,在没有MATLAB软件和许可证的计算机上无法访问。


c、 我们发现,最好每张幻灯片至少使用10张图片,每个基因型/条件使用3张幻灯片(幼虫)。


d、 为了比较条件/基因型之间相关系数的分布,使用以下方法对相关系数r进行Fisher z变换:


 


z=0.5英寸[(1+r)/(1-r)]


 


然后可以使用t检验或单因素方差分析比较转换后的相关系数z的分布。如果使用Prism,则使用Tukey的多重比较后验,单向方差分析对这些3+条件/基因型的数据是一种稳健的统计分析。




食谱


 


1.     冷泉港实验室配方


试剂


1x金额


最终1x浓度


10倍库存金额


最终10倍浓度


氯化钠


8克


137毫米


80克


37.1米


氯化钾


0.2克


2.7毫米


2克


27毫米


Na2HPO4


1.44克


10毫米


14.4克


100毫米


KH2PO4


0.24克


1.8毫米


2.4克


18毫米


注:1倍HCl时pH值为7.4。


2.     PBST公司


PBS+0.1%吐温20,从20%的储存溶液中稀释


3.     固定液:2%PFA/45%醋酸


16%PFA在Milli-Q H2O中稀释至2%和100%乙酸稀释至45%


**每解剖30分钟新鲜制备固定液


***16%的PFA在开瓶后4℃保存,3周内使用或染色体结构保存会受损


4.     45%醋酸


用Milli-Q H2O稀释至45%的100%乙酸


**每30分钟解剖一次


5.     阻断液/抗体鸡尾酒碱


PBS+0.1%吐温20+3%BSA(BSA稀释度为每体积的质量,即克每毫升)


 


致谢


 


该方案最初发表在库恩等人的研究手稿中。(2019年)。我们感谢Capelson和Little labs的成员在推动该方法发展的研究项目上的投入,并感谢J.Aleman在照片收集方面的帮助。M、 C.得到了美国癌症协会研究学者Grant RSG-15-159-01-CSM和NIH R01GM124143的支持。


 


相互竞争的利益


 


作者声明不存在利益冲突。


 


工具书类


 


1.     Cai,W.,Jin,Y.,Girton,J.,Johansen,J.和Johansen,K.M.(2010年)。用于抗体标记的果蝇多线染色体压片的制备。视觉实验(36)。内政部:10.3791/1748。


2.     Kuhn,T.M.,Pascual Garcia,P.,Gozalo,A.,Little,S.C.和Capelson,M.(2019年)。核孔蛋白的染色质靶向诱导染色质去浓缩。细胞生物学杂志218(9):2945-2961。


3.     帕罗,R.(2008年)。免疫染色法定位多线染色体上的蛋白质分布。2008年CSH协议:pdb.prot4714型.


4.     Capelson,M.,Liang,Y.,Schulte,R.,Mair,W.,Wagner,U.和Hetzer,M.W.(2010年)。染色质结合的核孔成分调节高等真核生物的基因表达。140室(3):372-383。


5.     Nakayama,T.,Shimojima,T.和Hirose,S.(2012年)。PBAP重构复合物是染色质边界组蛋白H3.3置换和边界功能所必需的。发展139(24):4582-4590。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kuhn, T. M., Little, S. C. and Capelson, M. (2020). Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations. Bio-protocol 10(13): e3673. DOI: 10.21769/BioProtoc.3673.
  2. Kuhn, T. M., Pascual-Garcia, P., Gozalo, A., Little, S. C. and Capelson, M. (2019). Chromatin targeting of nuclear pore proteins induces chromatin decondensation. J Cell Biol 218(9): 2945-2961.
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