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2020

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A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
用常规和体积电子显微镜对秀丽隐杆线虫胚胎进行超微结构分析的高压冷冻和冷冻替代的工作流程   

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Abstract

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote (Rahman et al., 2020).

Keywords: High-pressure freezing (高压冷冻), Freeze substitution (冷冻置换), C. elegans (秀丽隐杆线虫), FIB-SEM (FIB-SEM), Volume electron microscopy (体积成像电子显微镜), vEM (vEM )

Background

C. elegans is a free-living nematode with many properties that make it amenable to scientific study: (1) the worms are ~1 mm long; (2) they are easy to grow, handle and maintain; (3) they proliferate rapidly and (4) they are amenable to genetic manipulations. The reader is encouraged to consult Corsi et al. (2015) for an excellent primer to C. elegans and its use as a model organism in biology. Embryonic cell division events in C. elegans are largely invariant both spatially and temporally, making the organism a robust eukaryotic model system (Oegema and Hyman, 2006). Transient changes in dynamic cellular components like the nuclear envelope and chromosomes during embryonic development can be well described at resolutions afforded by fluorescence microscopy (Cohen-Fix and Askjaer, 2017), but capturing the corresponding ultrastructural changes at higher resolutions, whether in two or three dimensions, is challenging (Altun et al., 2002). The chitinaceous shell of the embryos poses a diffusion barrier to chemicals, precluding conventional aldehyde-based fixation protocols; thus, rapid freezing of samples followed by freeze-substitution and imaging by EM is the preferred way to capture ultrastructural intermediates at nanometer resolutions. As these samples are too large for simple plunge freezing, they have to be high-pressure frozen, ideally in a manner conducive to screening for the correct developmental stage (Muller-Reichert et al., 2003; McDonald et al., 2010). Recently, we published a report describing architectural intermediates in nuclear envelope breakdown during embryonic development (Rahman et al., 2020). We visually followed C. elegans embryos trapped in capillaries until just before high-pressure freezing to ensure that the correct stages were frozen. In an accompanying methods paper, we also reported cryo-fluorescence microscopy of high-pressure frozen whole C. elegans worms followed by correlative FIB-SEM (Chang et al., 2020) to image such structures in the intact worm. In both these advances, central experimental steps included the appropriate high-pressure freezing, freeze-substitution and resin embedding of trapped worms and/or embryos. Here we provide a step-by-step protocol to correctly execute these procedures for downstream vEM analysis, either to replicate our findings, or to answer other questions of interest in C. elegans.


Materials and Reagents

  1. C. elegans maintenance

    1. Tissue culture dish, 35 × 10 mm (Corning, Falcon, catalog number: 353001)

    2. Worm pick (Genesee Scientific, catalog number: 59-30P16)

    3. Micropipette tips with barrier (any brand, 1 µl, 20 µl and 1,000 µl)

    4. Serological pipets, 10 ml for OP50 seeding (Corning, Falcon, catalog number: 357551)

    5. C. elegans Bristol N2 [Caenorhabditis Genetics Center (CGC), https://cgc.umn.edu (Stiernagle, 2006)]

    6. E. coli OP50 strain DA735 [Caenorhabditis Genetics Center (CGC), https://cgc.umn.edu]

    7. Water, molecular biology grade (GE Healthcare, HyClone, catalog number: SH30538.02)

    8. Agar (RPI, catalog number: A20020-5000)

    9. Yeast extract (ThermoFisher Scientific, catalog number: BP9727-2)

    10. Bacto Peptone (BD, Bacto, catalog number: 211677)

    11. Sodium chloride (Avantor Performance Materials, J.T. Baker, catalog number: 3624-05)

    12. Cholesterol (Sigma, catalog number: 1580-01)

    13. Ethanol 200 proof (Decon Labs, catalog number: 2716)

    14. Trizma-Cl (Roche, catalog number: 10812846001)

    15. Trizma-OH (Roche, catalog number:10708976001)

    16. Luria-Bertani broth, bacterial culture medium (KD medical, catalog number: BLC-5020)

    17. M9 buffer (IPM Scientific USA, catalog number: 11006-517)

    18. Levamisole hydrochloride (Sigma, catalog number: 196142)


  2. High-pressure freezing and freeze substitution

    1. Glass microscopy slides (ThermoFisher Scientific, catalog number: 10144633B)

    2. Tubes, 1.5 ml for BSA solution aliquots (ThermoFisher Scientific, catalog number: 05-408-129)

    3. Micropipette tips with barrier (any brand, 1 µl, 20 µl and 1,000 µl)

    4. Micropipette tips without barrier (Eppendorf, 1 µl, catalog number: 30072.014)

    5. Cellulose capillary tube (Leica, catalog number: 16706869)

    6. Needles, 21-gauge (G) x 1½ inch (Covidien Monoject, catalog number: 305167)

    7. Syringe, disposable (VWR, EMS, catalog number: 72508 for 2.5 ml or 72509 for 5 ml)

    8. Alcohol swabs (BD medical, catalog number: 326895)

    9. Type A gold-coated copper planchette (Leica, catalog number: 16770152)

    10. Type B gold-coated copper planchette (Leica, catalog number: 16770153)

    11. Cryomarker, Black (ThermoFisher Scientific, Nalgene, catalog number: 22-026-700)

    12. Sample holder Cartridge system, D3 mm half cylinder (Leica, catalog number: 771849)

    13. Sample holder Cartridge system, D3 mm middle plate (Leica, catalog number: 771813)

    14. Nalgene cryovials (ThermoFisher Scientific, catalog number: 5000-1012)

    15. Polypropylene tubes, 50 ml (BD Falcon, catalog number: 352098)

    16. String or twine (VWR, Twine, catalog number: 30-33113)

    17. Disposable cellulose acetate filter, 0.22 µm mesh size, diameter 25 mm (Millipore, Cameo syringe filter, catalog number: 1213657)

    18. Erlenmeyer glass flasks, 250 ml (VWR, Pyrex, catalog number: 4444-250)

    19. Pasteur pipettes, Borosilicate glass, 5 ml (ThermoFisher Scientific, catalog number: 13-678-20A)

    20. Pasteur pipettes, plastic, 2 ml and 7 ml (Globe Scientific, catalog numbers: 137040 and 134090)

    21. Serological pipette, Borosilicate glass, 1 ml (VWR, catalog number: 93000-682)

    22. Plastic cups (for weighing resin) (VWR, Therapak, catalog number: 74850)

    23. Beem capsules (Ted Pella, catalog number: 69910-01)

    24. Beem capsule holder (Ted Pella, catalog number: 132-B)

    25. Double Edge Carbon steel blade (Ted Pella, Feather, catalog number: 121-9)

    26. Kimwipes 05517 (ThermoFisher Scientific, Kimberly-Clark Kimtech Science Precision wipes, catalog number: 06-677-72)

    27. Styrofoam container or tray (e.g., a shipping container lid, typically 15 × 20 cm and 2 cm deep)

    28. Disposable polypropylene spatula for osmium handling (VWR, catalog number: 80081-194)

    29. Bovine Serum Albumin (BSA), heat shock fraction (Sigma, catalog number: A3294)

    30. Nail polish (any color)

    31. Osmium tetroxide (OsO4) granules (EMS, catalog number: 19134)

    32. Acetone (ThermoFisher Scientific, catalog number: 9011)

    33. Uranyl acetate (EMS, catalog number: 22400)

    34. Methanol (Mallinckrodt, catalog number: 3016)

    35. Poly/Bed 812 embedding kit with DMP-30 (Polysciences, catalog numbers: 08792 and 08791)

    36. Dry ice (in-house supply)

    37. Double deionized water (in-house supply)

    38. Dry liquid nitrogen (LN2) (in-house supply)

    39. Dodecenyl succinic anhydride (DDSA) (Polysciences, catalog number: 08792, kit same as 35)

    40. Nadic Methyl anhydride (NMA) (Polysciences, catalog number: 08792, kit same as 35)

    41. Modified Youngren’s, Only Bacto-peptone (MYOB) plates for worm maintenance (see Recipes)

    42. Cholesterol stock solution (see Recipes)

    43. E. coli OP50 stock (see Recipes)

    44. Cellulose capillary attachment (see Recipes)

    45. 20% BSA solution (see Recipes)

    46. 25 mM Levamisole solution (see Recipes)

    47. Quick Freeze-substitution (QFS) cocktail (see Recipes)

    48. Poly/Bed 812 resin mix (see Recipes)

Equipment

  1. Micropipettes (any brand, for 1 µl, 20 µl and 1,000 µl volumes)

  2. Sharp-point tweezers (EMS, Dumont, catalog numbers: 78320-51T, 72919-0A, and 78340-51S) (Figure 1a-1c)

  3. Tweezers insulated with PVC (EMS, Dumont, catalog number: 3C-linox-E) (Figure 1d)

  4. Forceps insulated with PVC, long (Leica, VOMM, catalog number: 22SAESD) (Figure 1e)

  5. Crimping tool, scalpel #20 (Bard-Parker, catalog number: 371620) (Figure 1f)



    Figure 1. Tools for sample handling. a-c. Sharp-point tweezers for sample handling (Section A Steps 4-5). d-e. Insulated tweezers for frozen sample recovery and transfer (Section B Steps 4-9, and Section C Steps 6-7). f. Crimping tool: #20 scalpel manually blunted by rubbing the blade gently on a metal surface (McDonald et al., 2010).


  6. Table-top centrifuge (Eppendorf, model: 5424 or similar)

  7. Bottom illuminated stereomicroscope with frosted glass stage (Leica, model: SM2745 or similar)

  8. Leica high-pressure freezer, ultra-low temperature equipped with stereomicroscope and funnel to fill LN2 (Leica, model: ICE)

  9. Frozen sample recovery cryobox (stainless steel tray, deep) with frozen sample release station and 3 mm punch, rod, and plug (Leica, Austria; EM ICE high-pressure freezer package)

  10. Mini benchtop orbital shaker (VWR, catalog number: 97109-890)

  11. Large surface slide warmer (Premiere, catalog number: XH-2002)

  12. Infrared thermometer (General Tools & Instruments, catalog number: IRT207)

  13. Analytical balance (Sartorious Corp, model: BCE64-15)

  14. Stirring plate (Corning, catalog number: PC420D or similar)

  15. Standard desiccator connected to a mechanical pump (Ted Pella, model: VRD4)

  16. Metal blocks with 12 mm holes (ThermoFisher Scientific, catalog number: 88880152)

  17. Dry ice buckets, multiple (VWR, Scienceware Magic Touch 2 with lid, catalog number: M16807-2001)

  18. Bunsen burner

  19. Chemical safety hood

  20. Laboratory timer

  21. Chemical scale (Mettler, model: AE240)

  22. Temperature-controlled oven (Quincy Lab, model: 20GC)

  23. Standard autoclave

  24. Hairdryer (any brand with hot air fan)

  25. Sample storage, large LN2 dewar (Taylor-Wharton, catalog number: HC34)

  26. Portable LN2 dewar on a rolling base, 25 L (Worthington, catalog number: LD25)

  27. Portable LN2 dewar, 4 L (Worthington, catalog number: LD4)

  28. PPE (lab coat, face shield and cold-resistant gloves) for handling LN2 (any brand)

  29. Facemask, surgical (Halyard health, catalog number: 28806; or any brand)

Procedure

  1. High-pressure freezing (HPF) of C. elegans individual embryos

  1. Start a worm culture by placing 20-30 starved L1 larva on a new MYOB agar plate seeded with E. coli OP50 bacteria (see Recipes). Maintain worms by transferring 2-3 adults to a new MYOB plate every fourth morning (Stiernagle, 2006).

  2. 72 h prior to the HPF experiment, transfer 5-6 gravid adults to each of several new MYOB plates (at least 3 separate plates to ensure enough young adults).

  3. In the morning of the HPF experiment, take out two aliquots (500 µl each) of 20% w/v BSA solution from 4 °C and spin at 94 x g (~1,000 rpm) for 5 min in a table-top centrifuge to remove bubbles. Keep at room temperature.

  4. Place 3-4 pairs of clean sharp-point tweezers (Figure 1a and 1b) next to the dissecting scope, and 1-2 pairs (Figure 1c) next to Leica ICE high-pressure freezer. Tweezers 1a and 1b are suitable for cellulose tube transfer while tweezer 1c is suitable for planchette transfer. It is critical to have alcohol swabs next to the tweezers (Figure 2a): unless you clean them frequently, the tweezers become sticky with 20% BSA solution, resulting in sample loss during transfer.

  5. Place one micropipette (1 µl) set at 0.8 µl and another micropipette (20 µl) set at 12 µl next to alcohol swab, tweezers, crimping tool (white arrow), and worm pick (yellow arrow) as in Figure 2a. Set up worm dissecting tool (syringe with 21G needles, Figure 2b). Place multiple cellulose tubes attached to pipette tips (Figure 2c, purple arrows and inset). Keep one micropipette (1 µl) dedicated for capillary tube handling (Figure 2c).

  6. Take out 9-10 planchettes (type A and type B separately) in 35 mm Petri dishes (Figure 2c), and mark type A cavities with a black marker to distinguish it from type B later.

    Note: Type A planchettes have a single 300 µm deep cavity on one face while the other face is flat; the samples will be placed later in the shallow cavity of a type B planchette (Step A14).



    Figure 2. Setup for worm dissection prior to HPF. a. Micropipettes (1 µl and 20 µl), sharp-point tweezers, alcohol swab, crimping tool (white arrow) and worm pick (yellow arrow). b. Worm dissecting tool – a pair of 21G needles on 2.5 ml syringe to be used as scissors to cut worms. c. A dedicated micropipette (1 µl) for handling cellulose capillaries attached to pipette tips (purple arrows and d). Planchettes type A and type B are placed in separate plastic Petri dishes. d. A cellulose capillary attachment (see Recipe 4) for embryo and/or worm collection.


  7. Check liquid nitrogen (LN2) transport dewars to make sure they are empty and dry. Fill one large dewar (25 L) and two medium dewars (4 L) with LN2.

  8. Turn on the Leica ICE high-pressure freezer (Figure 3a) and wait to hear the compressor turn on (identified by a hum and mild vibration), which should also be reflected in the monitor status screen. The loading station (Figure 3b) should be clean and dry. Fill the freezer chamber (Figure 3c) with LN2 from the 25 L dewar slowly with multiple brief pauses to avoid triggering the alarm and a false indication that the tank is full. A significant amount of LN2 evaporates in the process of chilling the tank.

    Note: You may need help from a second person to pour ~18 liters of LN2 safely.

  9. Assemble HPF sample storage dewar (Figures 3d-3f). Note the trisection pod in the dewar can hold up to three cartridge systems in each of three cups, equating to a maximum of nine HPF “shots” before the sample storage dewar is full. The high-pressure freezer requires ~20 min to equilibrate to LN2 temperature; once equilibrated, fill the HPF sample storage dewar (Figure 3f) with LN2 and insert into Leica ICE freezer chamber (Figure 3g, underneath the loading station in Figure 3a). You must wait until the freezer is ready, otherwise the container will collect and freeze condensation from the air forming ice crystals. Crystalline ice or frost must be avoided in this procedure, as with other cryogenic experiments. So, maintain dry conditions, and work at a quick pace to minimize exposure of the sample and tools to humidity.

  10. Assemble plastic adapters on the two halves of the Leica ICE high-pressure freezer loading station (Figures 3h-3j, and arrows in Figure 3b). Place the planchette holder with a hole in it (Figure 3i) on top of a plastic adapter placed on the bottom steel surface (Figure 3b, yellow arrow). Run a blank HPF cycle by manually closing the red lid (Figure 3b) to ensure the freezer is working properly (Figure 5).



    Figure 3. Setting up the Leica EM ICE for HPF. a. Leica EM ICE high-pressure freezer. b. Loading station, enlarged from boxed area in 3a. Arrows show where planchette holders are placed for each HPF run, with a half cylinder on top (white arrow and 3h), and planchette holder with a hole in it (3i) atop the other half cylinder placed on the bottom black steel half (yellow arrow and 3j). c. LN2 filling port behind grey door (long black arrow). d-f. Assembly of HPF sample storage dewar comprising the trisection pod (3d) and segmenting insert (grey cylinders in 3e). Insets in 3e show top view after assembly; blue arrow, “bayonet” or release button and yellow arrowhead, lock/open position. The bayonet is pressed and rotated counter-clockwise or clockwise to open or lock respectively (left inset – bayonet at open position, and right inset – bayonet at close position). g. Collection holder unit inserted into receptacle before automatic withdrawal into the freezer unit, seen as a black outlined panel below the loading station in 3a. The notch with the “in >” label (3e inset, yellow ring) should face the HPF instrument holding unit. h-j. Leica cartridge system contains two identical half cylinders and a planchette holder with a hole in it.


  11. Collect 3-4 young adult worms with a worm pick and place in a drop (12 µl) of 20% BSA solution on a clean glass slide. Use two needles as shears (Figure 4a) to cut the worms open (at the middle of the body) to release the embryos (McDonald, 1999; Muller-Reichert et al., 2007; McDonald et al., 2010).

    Note: If whole worms are collected, use 20% BSA solution with 25 mM levamisole and incubate 2-3 min to anesthetize the worms. The worms can be taken up into capillaries individually, as below.

  12. Visually scan the embryos to find one at the desired stage of embryonic development, keeping in mind that the steps leading to HPF will take approximately 2 min. Avoid prolonged observation under the dissecting scope as the small volume of 20% BSA solution will quickly dry out due to evaporation.

  13. To collect an embryo of desired stage, place the open end of a cellulose capillary tube, attached to pipette tip (Figure 4b, cartoon) close to the embryo. Due to capillary action, the embryo will get into the capillary tube and there is no need to pipette it in (Figure 4c, cartoon).

  14. Use a crimping tool to press gently on the capillary tube (otherwise it will cut open and embryo will float away) on both sides of the trapped embryo to seal the capillary tube (refer to Figure 4c, cartoon). Use the same tool to separate the section (Figure 4c, inset) with trapped embryo away from dissected worms. Remember to keep the length of this section around or below 2 mm (Figure 4c, inset) so that it will fit in the cavity of the type B planchette (Muller-Reichert et al., 2008). Follow embryonic development of the trapped embryo under a dissecting scope.

  15. Approximately 1 min prior to the desired embryonic developmental stage, add 0.8 µl of 20% BSA solution into the 100 µm cavity of the type B planchette (Figure 4d). The planchette must be filled to the top to avoid air bubbles, but not overfilled to avoid excessive liquid wicking into the loading area. A gentle positive meniscus typically suffices. Transfer the capillary tube section with the embryo (Figure 4c, inset) with sharp-point tweezers into the type B planchette filled with 20% BSA solution (for ease of visualization, capillary tube sections with intact worms are shown in Figure 4d). Place a type A planchette (flat side) on top of the type B planchette (Figure 4g).

  16. Transfer the planchette sandwitch with the embryo secured inside (Figures 4e-g) to the Leica EM ICE loading station (Figure 3d, pre-assembled cartridege system; also see Figures 4h-i). Manually closing the red flap (Figure 4j) will initiate the freezing process, where the embryo secured inside the cartridge system is cooled to LN2 temperatures within tens of milliseconds and under high pressures of approximately 2,000 bar (refer to Figure 5).



    Figure 4. Sample preparation for HPF. a-c. Dissection of young adult worms in a drop of 20% BSA solution. Steps are depicted in a cartoon above each image. An embryo of choice is collected into a cellulose capillary and trapped with the crimping tool (c, inset). d. Planchette with whole worms inside cellulose capillaries in 20% BSA solution filled to the top. e-g. A sharp-point tweezer which is used to transfer the capillary piece (c, inset) to the type B planchette cavity, is used to place the type A planchette (flat side) on top of type B planchette. h-j. The planchette sandwich (g, right) is placed securely in the holder (h, orange arrow). Manually closing the red lid (j) on the loading station will plunge the sample into LN2 under high pressure.



    Figure 5. Temperature and pressure plot of a high-pressure freezing cycle. The x axis is time in milliseconds (ms), and the y axis is either pressure in bar, red, or temperature in Kelvin (K), blue. For only illustration purpose, here is a screenshot of an acceptable execution of a single run from the high-pressure freezer, with pressure reading of 2237 bar at time sample cooling started at a rate (dT/dt) of 18163 K/s. Using the zoom out button (right bottom corner) pressure, the temperature status can be obtained for up to 600 ms (see inset). For details refer to Muller-Reichert et al. (2007 and 2008).


  17. Repeat the Steps A10 to A15 until you have frozen the necessary number of samples. The maximum capacity of a single sample storage dewar in the Leica ICE high-pressure freezer is nine samples, however a blank HPF run is typically executed at the start of the experiment, leaving space for up to eight samples. Note that it is possible to switch the sample collection to a new and dry holder (Figure 3d) to collect another set of up to nine samples. Other high-pressure freezers do not have a limit of nine samples.

  18. High-pressure frozen samples can be stored under LN2 for years. For long-term storage of samples go to protocol B. For freeze substitution, go to protocol C directly, skipping protocol B.


  1. Recovery of frozen samples from HPF machine

  1. To store frozen samples, prepare a 50 ml polypropylene tube by perforating its side wall with a hot end of a sharp mini screwdriver (place the tip on a Bunsen burner briefly to heat). The hole prevents pressure build up and a potential explosion hazard in case of accidental warming. Attach a long (~3 feet) string securely in the cap (Figure 6) and a tape label so the tube holding frozen samples can be pulled out of a large LN2 tank.



    Figure 6. Preparation of storage tube. A 50 ml blue cap tube is prepared as described in Section B Step 1. Inset: An example of label on the tube and tape label to the string.


  2. Prepare multiple 1 ml cryovials by perforating its side wall (similar to Step B1). The hole prevents pressure buildup in the tube. Label each cryovial with date, experiment number and sample number using a pencil. Pencil marks are stable in acetone and help prevent mix-up later.

  3. Use a hairdryer to remove moisture completely from the sample unloading chamber and accessory units (Figure 7a). Fill the chamber slowly with LN2, and wait until there are no bubbles, that is, the LN2 surface is calm. Keep the plexiglass cover closed to minimize ice or frost contamination.

  4. Place all tweezers and other accessories on a slide warmer set at 45 °C next to the chamber and keep the hairdryer plugged in as you will need it again. Be quick and alternate the tweezers kept on a slide warmer to prevent depositing ice crystals into sample unloading chamber.

    Note: Wearing a surgical facemask reduces condensation and fog in chamber filled with LN2.

  5. Take the sample storage dewar out from Leica ICE high-pressure freezer. Transfer the segmenting insert and attached trisection pod (Figure 7c) to the designated slot (Figure 7a, left side) in the cryobox and release the trisection pod containing the planchette assemblies (Figures 7c-7d) by pressing and twisting the “bayonet” or release button from the locked to open position on the segmenting insert (Figure 7c, blue arrow).

  6. Dip a dry insulated tweezer (Figure 1d) in LN2 away from the samples for about 20 s to chill it to LN2 temperature. Watch for the bubbles and wait until the hissing sound ceases.

  7. Remove plastic adapters one at a time out of the sample holder (Figure 7e) leaving only the holder with frozen planchettes. Quickly transfer the holder with frozen planchettes (keeping submerged in LN2 all the time) to the sample release station (Figures 7e-7f, right side).

  8. With the release handle (Figure 7a, white arrow), punch out the frozen planchettes from the holder into the reservoir (Figure 7g, yellow arrow). Keep the plexiglass cover closed to minimize ice contamination when not transferring/releasing samples.

  9. Place prelabeled cryovials into the cryobox and allow to chill to LN2 temperature for a few minutes. Use dry tweezers (Figure 1d) to transfer frozen planchettes into respective cryovials (keeping submerged in LN2 all the time). Warm up each cryovial cap with your palm, and then quickly close. If the cap is not warm and moisture free, then it will freeze to the tube and it is nearly impossible to reopen the tube without warming up the whole tube, causing damage to the frozen sample.



    Figure 7. Sample recovery after high-pressure freezing. All photographs were taken without LN2 for clarity. a. Sample recovery cryobox showing a tray for sample holder (left) and sample release station with release handle (white arrow) and collection reservoir on the right. b-d. Sample holder from ICE high-pressure freezer transferred to sample recovery cryobox. Twisting the bayonet (c, blue arrow) counter clockwise releases the sample collection container unit from the cylinders. e-g. One sample holder at a time is transferred to the release station. Frozen planchettes are collected into the reservoir (g, orange arrow).


  10. Final Check: Inspect for any split planchettes, and if found, collect the ones without the black dot. The type-A planchette (flat bottom) used as a cover is marked with a black dot, and can be easily identified under LN2 and discarded, as the frozen embryo is inside the cavity of an unmarked, type B planchette.

  11. Collect all cryovials into a prelabeled 50 ml polypropylene tube (prechilled in LN2 in a portable dewar), close it with a cap with the string with tape label visible outside (see Figure 6), and deposit into a LN2 storage tank. Samples can be stored for years with appropriate care.


  1. Freeze substitution of HPF embryos for electron microscopy

Note: This process takes 3-4 days in total and likely more than 8 h on the first day due to long incubation times. Confirm that you have enough dry ice and LN2 supply. Reagents must be freshly prepared, so plan your day accordingly. Use a slide warmer and hairdryer to keep all instruments moisture free!

  1. Prepare the QFS cocktail as per recipe 7 (Rahman et al., 2020), and aliquot 1 ml each in prelabeled cryovials (label as per sample number, one frozen sample per vial).

  2. Place a metal block with 12 mm holes facing up in dry ice bucket. Fill with LN2 and allow to chill to LN2 temperature. Add LN2 frequently to keep the block submerged. Place a lid on the bucket to prevent condensation. The bucket must be placed in a chemical fume hood, and subsequent steps performed in a fume hood as osmium is toxic and volatile.

  3. Freeze the QFS cocktail by placing the tubes into the metal block submerged in LN2. Do not add too much LN2, otherwise the tubes will float out of the block.

  4. Collect the sample cryovials (HPF embryos or worms) from the LN2 storage tank into a transfer dewar filled with LN2 (Figure 8a, blue arrow).



    Figure 8. Sample transfer for QFS. a. Frozen samples are transferred in LN2 dewar (bottom, blue arrow). QFS cocktail submerged in LN2 in a dry ice bucket (top left, yellow arrow). b. A shallow Styrofoam container with frozen QFS cocktail cryovial (top left) and HPF sample cryovial (top right) laid flat submerged in LN2. One tube at a time, cryovials are opened (top right), frozen planchettes are spilled into LN2 on a shallow Styrofoam container, and then transferred into frozen QFS cocktail in cryovials laid flat on the same surface (top left) as in Steps C5 to C7 with tweezers. c. QFS tubes are placed on dry ice sideways (in a prechilled metal block). d. The bucket is filled with more dry ice, covered with a lid, and then placed on an orbital shaker (McDonald and Webb, 2011).


  5. Take a clean and dry shallow Styrofoam container or tray. A lid of a shipping container, typically 15 × 20 cm and 2 cm deep, will suffice. Add LN2 to the tray and allow it to cool just prior to the subsequent steps.

  6. Take out one cryovial at a time. Uncap the cryovial, and quickly lay down the vial flat on a shallow Styrofoam container filled with LN2 and spill out the planchette under LN2. Next to it also uncap and lay down a frozen QFS-containing vial flat in the LN2. Keep the cap of this vial warm in your palm or on a slide warmer to avoid condensation otherwise it will freeze and get stuck.

  7. Transfer one planchette into each vial with frozen QFS cocktail with a sharp-point tweezer. Quickly stand up the cryovial (keeping most of the vial submerged in LN2) and screw the warm cap onto the tube. Immediately transfer it to the metal block submerged in LN2 (Step C2). It is possible to place multiple planchettes in a single QFS cocktail tube if samples are easily identifiable, however we warn that the planchettes can jostle against each other and dislodge large samples during QFS.

  8. Frequently check and add LN2 to keep the metal block submerged the whole time. Place the lid on top to restrict condensation (as much as possible) from the moisture in the air.

  9. Repeat Steps C4 to C7 until all sample-containing planchettes are transferred to individually labeled QFS-containing cryovials. We advise not to attempt more than eight tubes at a time.

  10. Carefully decant LN2 and fill the ice bucket halfway with dry ice. Rotate the metal block so cryovials are now lying flat on their side. Add more dry ice to cover the metal block completely and cover the bucket with a lid. Place it on an orbital shaker set at 60 cycles/min for 3 h inside a chemical safety hood (McDonald and Webb, 2011).

  11. Discard dry ice after 3 h. Place the lid back and continue to rotate at 60 cycles/min for another hour.

  12. Remove the lid and continue to shake at 60 cycles/min for another hour (or as needed) until the metal block reaches a temperature of about 4 °C. Frequently check the temperature of the block with an infrared thermometer. This is a critical step! Long exposure at 4 °C or higher may significantly darken the entire volume, making it hard to identify a single embryo inside the capillary.

  13. Replace the QFS cocktail with 100% acetone (stored at 4 °C). First, carefully remove the QFS cocktail with a glass pipette (take care to leave the planchette undisturbed). Use a fresh pipette, and gently refill by releasing the solution (100% acetone) against the side wall of the cryovial. Replace the solution with the following mixture after 1 h incubation in each (use an orbital shaker set at 60 cycles/min).

    1. 1:2 resin:acetone

    2. 1:1 resin:acetone

    3. 2:1 resin:acetone

      Discard all pipettes, used and unused reagents, paper towels and kimwipes used in the process in accordance with institutional biohazard disposal procedure.

  14. Transfer the planchettes into 100% resin and leave overnight (14 h to 16 h) at room temperature on an orbital shaker set at 60 cycles/min.

  15. Next morning, replace overnight resin with freshly prepared 100% resin and immediately proceed to the next step.

  16. Use a razor blade to cut off the bottom of a Beem capsule; uncap the capsule and flex the hinge so that the lid lies flat on a surface. Place the planchette sample side up along with a few drops of resin on the inside of the Beem capsule lid. Carefully close the capsule onto the lid, keeping the planchette sample side up. The bottom of the capsule with the cut-out hole should be facing up. Fill the capsule through this opening with 100% resin (see Figures 9a-9i), and bake it to cure in an oven at 75 °C for 60 h to 65 h.

  17. Once cured, use a razor to cut off the plastic beam capsule (see Figures 9j-9m). Now carefully remove all the resin around the metal planchette under a dissecting scope. Important: be sure to completely remove all resin on the sides – the shiny metal of the planchette side should be totally exposed (see Figures 9o-9p). Now quickly immerse the exposed metal base of the planchette into LN2 until it reaches cryogenic temperature, then heat with a hairdryer until all condensation has disappeared and the planchette is warm. Repeat the cold/hot cycle multiple times as required, until the planchette or the metal carrier pops out and falls away due to differential thermal expansion between the metal and resin. This will leave the resin-embedded sample intact (Figure 9r). Do not use force to pry the planchette loose, as this risks fracturing the resin leaving the resin-embedded sample still in the cavity. You can strore resin-embedded samples either with or without planchette attached for years in a dustproof storage container at room temperature before sectioning for imaging (TEM or vEM).


    Note: The QFS protocol described here is adequate for FIB-SEM imaging and array tomography in our hands. For conventional TEM imaging, the samples may be sectioned and are typically post-stained to enhance contrast (Hayat, 2000). For other volume EM approaches such as serial block face SEM, users may wish to further enhance metallization of the sample for high-resolution work by adapting room temperature en-bloc staining protocols (Hayat and Giaquinta, 1970; Hua et al., 2015).



    Figure 9. Resin embedding and sample processing. a-c. A razor blade is used to cut off the bottom of a Beem capsule. d-e. Flex the hinge so that the lid lies flat on surface. Place the planchette (from QFS Step C15) sample side up and add a few drops of resin with a Pasteur pipette. f-g. Carefully close the capsule on the lid keeping sample side of the planchette up. h-i. Fill the Beem capsule with 100% resin and transfer to an oven (see Step C16) to cure. After curing, the stained sample is at the bottom of the capsule (9j, back view). k-m. Use a razor blade to remove the Beem capsule. n-p. Under a dissecting scope carefully remove the resin using a razor blade to expose the planchette. q. Once the planchette is exposed multiple times, immerse it in LN2 followed by heating with a hairdryer until the metal carrier pops out. r. Exposed sample embedded in 100% resin after removal of planchette or metal carrier.

Notes

Working with liquid nitrogen is potentially dangerous. Appropriate Personal Protective Equipment (PPE) must be used to protect your eyes, face and exposed skin. Also see your institute’s safety procedure.

  Osmium, a heavy metal, poses health risk if inhaled. You must wear a surgical mask and prepare the freeze substitution cocktail mix in a chemical hood connected to an exhaust system.

Recipes

  1. Modified Youngren’s, Only Bacto-peptone (MYOB) plates for worm maintenance

    Bacto Agar 20 g

    Sodium chloride (NaCl) 2 g

    Trizma-HCl 0.55 g

    Trizma-OH 0.24 g

    Bacto Peptone 3.1 g

    Deionized water to 1 liter

    1. Autoclave for 20 min (liquid cycle), allow to cool down

    2. Add 1.6 ml cholesterol from stock solution (see below)

    3. Mix thoroughly with a magnetic stirrer prior to pouring into 35 mm tissue culture dishes, about 5 ml per plate (~200 plates per liter)

    4. Once the agar plates have solidified and are dry, apply ~200 µl E. coli OP50 culture (see below) and let stand for two days at room temperature until the bacterial solution has dried and a bacterial lawn is formed (Church et al., 1995)

  2. Cholesterol stock solution

    Cholesterol 5 mg

    Ethanol (200 proof) 100 ml

    Use a small magnetic stir bar to dissolve cholesterol (slow speed for about 2 h)

  3. E. coli OP50 stock

    From a frozen stock, streak bacteria on an agar plate (without any antibiotics) and incubate overnight (~14-16 h) at 37 °C. Inoculate a few isolated colonies into 100 ml LB media (without any antibiotics) and incubate for 6-7 h at 37 °C without shaking. Seed MYOB plates with 200 µl culture (Stiernagle, 2006). Dispose of unused bacterial culture appropriately.

  4. Cellulose capillary attachment

    See Figure 10



    Figure 10. Cellulose capillary attachment for sample collection. Cut cellulose capillary into ~4 cm pieces using sharp scissors. Under a dissecting scope, push or pull the capillary through a 1 ul pipette tip (without a barrier) until the edge of the capillary tube is close to the edge of the tip. Apply nail polish to glue the capillary tube to the tip of the pipette tip and allow it to dry at room temperature for an hour (Muller-Reichert et al., 2003). Ensure the other end of the capillary tube is open; if not, cut to open near the edge with sharp scissors (see arrowhead).


  5. 20% BSA solution

    BSA 10 g

    M9 buffer to 50 ml

    1. Prepare the solution in multiple steps, that is, each time add a small amount of BSA into 35 ml of M9 buffer (in a 50 ml blue cap tube)

    2. Mix gently on a table-top orbital shaker at low speed to avoid generating bubbles

    3. Once BSA is completely dissolved, bring volume to 50 ml. Mix gently to homogeneity and store at 4 °C, or -20 °C in small aliquots (long-term preservation) for up to one year

  6. 25 mM Levamisole solution

    1. Add 0.051 g levamisole into 10 ml of 20% BSA solution

    2. Mix gently on a table-top orbital shaker at low speed to avoid generating bubbles

    Note: The solution can be used for up to two to three months.

  7. Quick Freeze-substitution (QFS) cocktail

    Note: Must prepare inside a chemical hood. Use a borosilicate glass pipette to measure acetone and methanol. Please follow institutional chemical safety regulations for handling Osmium and Uranium compounds. These are extremely toxic; safety is paramount.

    OsO4 granule 0.1 g

    Acetone 13.5 ml

    At room temperature stir to mix completely for about 10-15 min. Once no residue is seen, add from a freshly opened vial of:

    2% Uranyl acetate (UA) in methanol 0.75 ml

    Distilled deionized (DD2) water 0.75 ml

    At room temperature mix completely and pass through a 0.22 µm cellulose acetate filter to remove any residual undissolved elements. Aliquot 1 ml into separate cryovials, flash freeze in LN2 and store at -80 °C (Rahman et al., 2020)

  8. Poly/Bed 812 resin mix

    Poly/Bed 812 14.6 g

    Dodecenyl succinic anhydride (DDSA) 8.4 g

    Nadic Methyl anhydride (NMA) 7.0 g

    DMP-30 0.42 ml

    Mix gently with magnetic stirrer until homogeneity is achieved. Prepare fresh from the kit which contains all the chemicals mentioned above (Rahman et al., 2020)

Acknowledgments

We thank Heather Berensmann for critical reading of the manuscript and help with photography.

    M.M. Rahman and O. Cohen-Fix were supported by the National Institute of Diabetes and Digestive and Kidney Disease (intramural grant DK069012). I.Y. Chang, and K. Narayan were supported by the National Cancer Institute (Contract No. 75N91019D00024). This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Competing interests

The authors declare no competing financial interests.

Ethics

Hazardous chemicals were disposed according to institutional guidelines.

References

  1. Altun, Z. F., Herndon, L. A., Wolkow, C. A., Crocker, C., Lints, R. and Hall, D. H. (Eds.) Worm Atlas, 2002-2020. 
  2. Chang, I. Y., Rahman, M. M., Harned, A., Cohen-Fix, O., and Narayan, K. (2020). Cryo-fluorescence microscopy of high-pressure frozen C. elegans enables correlative FIB-SEM imaging of targeted embryonic stages in the intact worm. Methods Cell Biol https://doi.org/10.1016/bs.mcb.2020.09.009.
  3. Church, D. L., Guan, K. L. and Lambie, E. J. (1995). Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans. Development 121(8): 2525-2535.
  4. Cohen-Fix, O. and Askjaer, P. (2017). Cell Biology of the Caenorhabditis elegans Nucleus. Genetics 205(1): 25-59.
  5. Corsi, A. K., Wightman, B. and Chalfie, M. (2015). A Transparent window into biology: A primer on Caenorhabditis elegans.WormBook 1-31. doi:10.1895/wormbook.1.177.1
  6. Hayat, M. A. (2000). Principles and techniques of electron microscopy: biological applications (4th edition). Cambridge, UK ; New York: Cambridge University Press.
  7. Hayat, M. A. and Giaquinta, R. (1970). Rapid fixation and embedding for electron microscopy. Tissue Cell 2(2): 191-195.
  8. Hua, Y., Laserstein, P. and Helmstaedter, M. (2015). Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6: 7923.
  9. McDonald, K. (1999). High-Pressure Freezing for Preservation of High Resolution Fine Structure and Antigenicity for Immunolabeling. In: Hajibagheri, N. (Ed.). Methods in Molecular Biology: Electron Microscopy Methods and Protocols (Vol. 117, pp. 77-97). Totowa, NJ: Humana Press Inc.
  10. McDonald, K. L. and Webb, R. I. (2011). Freeze substitution in 3 hours or less. J Microsc 243(3): 227-233.
  11. McDonald, K., Schwarz, H., Muller-Reichert, T., Webb, R., Buser, C. and Morphew, M. (2010). "Tips and tricks" for high-pressure freezing of model systems. Methods Cell Biol 96: 671-693.
  12. Muller-Reichert, T., Hohenberg, H., O'Toole, E. T. and McDonald, K. (2003). Cryoimmobilization and three-dimensional visualization of C. elegans ultrastructure. J Microsc 212(Pt 1): 71-80.
  13. Muller-Reichert, T., Mantler, J., Srayko, M. and O'Toole, E. (2008). Electron microscopy of the early Caenorhabditis elegans embryo. J Microsc 230(Pt 2): 297-307.
  14. Muller-Reichert, T., Srayko, M., Hyman, A., O'Toole, E. T. and McDonald, K. (2007). Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis. Methods Cell Biol 79: 101-119.
  15. Oegema, K. and Hyman, A. A. (2006). Cell division. WormBook 1-40. doi:10.1895/wormbook.1.72.1
  16. Rahman, M., Chang, I. Y., Harned, A., Maheshwari, R., Amoateng, K., Narayan, K. and Cohen-Fix, O. (2020). C. elegans pronuclei fuse after fertilization through a novel membrane structure. J Cell Biol 219(2). doi:10.1083/jcb.201909137.
  17. Stiernagle, T. (2006). Maintenance of C. elegans. WormBook 1-11. doi:10.1895/wormbook.1.101.1.


简介

[摘要]自由生活的线虫秀丽隐杆线虫是研究发育生物学的流行模型系统。在这里,我们描述了详细的协议,以高压冷冻线虫的胚胎(解剖后离体,或完整的蠕虫内),然后快速冷冻替代。经过处理的样品适合通过常规电子显微镜(EM)或更新的体积EM(vEM)方法(如Focuse d离子束扫描电子显微镜(FIB-SEM))进行超微结构分析。的细胞特征,例如超微结构的NUCL耳信封,染色体,内质网和线粒体保存良好这些实验程序后,并产生精确的三维模型用于可视化和分析(张等人,2020)。在秀丽隐杆线虫合子的前核相遇后,该方案被用于膜和染色体的3D重建(Rahman等,2020)。

[背景技术]线虫是自由生活线虫具有许多特性,使其适合于科学的研究:(1)将蠕虫是〜1毫米长; (2)它们易于生长,处理和维护;(3)它们迅速增殖,并且(4)它们适合进行基因操作。鼓励读者咨询科希等人。(2015年),为秀丽隐杆线虫及其在生物学中作为模型生物的优秀引物。Ë在mbryonic细胞分裂事件线虫在很大程度上是不变的空间和时间,使机体强大的真核模型系统(Oegema和海曼,2006年)。胚胎发育过程中动态细胞成分(如核被膜和染色体)的瞬态变化可以用荧光显微镜提供的分辨率很好地描述(Cohen-Fix和Askjaer,2017),但是可以以更高的分辨率捕获相应的超微结构变化,无论是在两个还是三个尺寸,是具有挑战性的(A升桶等人,2002) 。个胚胎电子chitinaceous壳构成的扩散阻挡层的化学品,排除常规醛系固定协议; 因此,快速冷冻样品,然后冷冻替代并通过EM成像是捕获纳米级分辨率的超微结构中间体的首选方法。由于这些样本太大,无法进行简单的急速冷冻,因此必须进行高压冷冻,理想情况下应采用有助于筛选正确发育阶段的方式(Muller-Reichert等,2003 ;McDonald等,2010 )。最近,我们发表了一份报告,描述了胚胎发育过程中核被膜破裂的建筑中间体(Rahman等,2020)。w ^ ê直观地追踪编线虫胚胎被困在毛细血管前才高压冻结,以确保正确的阶段被冻结。在随附的方法文件中,我们还报道了高压冷冻的整个秀丽隐杆线虫蠕虫的冷冻荧光显微镜检查,然后进行了相关的FIB-SEM(Chang等人,2020年)以对完整蠕虫中的此类结构进行成像。在这两项进展中,主要的实验步骤包括对捕获的蠕虫和/或胚胎进行适当的高压冷冻,冷冻替代和树脂包埋。在这里,我们提供了一个分步协议,可以正确执行这些过程以进行下游vEM分析,以复制我们的发现,或回答秀丽隐杆线虫的其他感兴趣的问题。

关键字:高压冷冻, 冷冻置换, 秀丽隐杆线虫, FIB-SEM, 体积成像电子显微镜, vEM



材料和试剂


秀丽隐杆线虫的维护
组织培养皿,35 × 10 mm(Corning,Falcon,货号:353001)
蠕虫镐(Genesee Scientific,目录号:59-30P16)
带挡板的微量移液器吸头(任何品牌,1 µl,20 µl和1,000 µl)
血清移液吸管,用于OP50接种的10 ml(Corning,Falcon,目录号:357551)
C. elegans Bristol N2 [秀丽隐杆线虫遗传学中心(CGC),https://cgc.umn.edu(Stiernagle,2006年)]
大肠杆菌OP50株DA735 [秀丽隐杆线虫遗传学中心(CGC),https: //cgc.umn.edu ]
水,分子生物学等级(GE Healthcare,HyClone,目录号:SH30538.02)
琼脂(RPI,目录号:A20020-5000)
酵母提取物(ThermoFisher Scientific,目录号:BP9727-2)
Bacto P蛋白one(BD,Bacto,目录号:211677)
氯化钠(Avantor Performance Materials,JT Baker,目录号:3624-05)
胆固醇(Sigma,目录号:1580-01)
乙醇200证明(Decon Labs,目录号:2716)
Trizma-Cl(罗氏(Roche),目录号:10812846001)
Trizma-OH(罗氏(Roche),目录号:10708976001)
Luria-Bertani肉汤,细菌培养基(KD medical,目录号:BLC-5020)
M9缓冲器(IPM Scientific USA,目录号:11006-517)
盐酸左旋咪唑(Sigma,目录号:196142)


高压冷冻和冷冻替代
玻璃显微镜载玻片(ThermoFisher Scientific,目录号:10144633B)
1.5毫升试管,用于BSA溶液等分试样(ThermoFisher Scientific,目录号:05-408-129)
具有阻隔微量提示(任何品牌,1微升,20微升和1 ,000微升)
微管TTE提示而不阻挡仪(Eppendorf,1微升,目录号:30072.014)
纤维素毛细管(徕卡,目录号:16706869)
21针(G)x 1针½英寸(Covidien Monoject,目录号:305167)
一次性注射器(VWR,EMS,目录号:2.5毫升为72508或5毫升为72509)
酒精拭子(BD医疗,目录号:326895)
A型镀金铜制手推车(Leica,目录号:16770152)
B型镀金铜制手推车(Leica,目录号:16770153)
黑色低温标记(ThermoFisher Scientific,Nalgene,目录号:22-026-700)
样品架药筒系统,D3 mm半圆筒(Leica,货号:771849)
样品架药筒系统,D3毫米中间板(Leica,货号:771813)
Nalgene冷冻管(ThermoFisher Scientific,目录号:5000-1012)
50毫升聚丙烯管(BD Falcon,目录号:352098)
细绳或细绳(VWR,细绳,货号:30-33113)
一次性醋酸纤维素过滤器,网目尺寸为0.22 µm,直径25 mm(Millipore,Cameo针筒式过滤器,目录号:1213657)
250毫升锥形玻璃烧瓶(VWR,Pyrex,目录号:4444-250)
巴斯德移液器,硼硅酸盐玻璃,5毫升(ThermoFisher Scientific,目录号:13-678-20A)
塑料巴斯德吸管,2 ml和7 ml(Globe Scientific,目录号:137040和134090)
血清移液器,硼硅酸盐玻璃杯,1毫升(VWR,目录号:93000-682)
塑料杯(用于称量树脂)(VWR,Therapak,目录号:74850)
Beem胶囊(Ted Pella,目录号:69910-01)
Beem胶囊固定器(Ted Pella,目录号:132-B)
双刃碳钢刀片(Ted Pella,羽毛,目录号:121-9)
Kimwipes 05517(ThermoFisher Scientific,Kimberly-Clark Kimtech Science精密湿巾,目录号:06-677-72)
发泡胶容器或托盘(例如,运输容器的盖子,通常15 × 20厘米,深2厘米)
用于处理os的一次性聚丙烯刮刀(VWR,目录号:80081-194)
牛血清白蛋白(BSA),热激分数(Sigma,目录号:A3294)
指甲油(任何颜色)
四氧化锇(OSO 4 )颗粒剂(EMS,Ç atalog号码:19134)
丙酮(赛默飞世科学,Ç atalog号:9011)
乙酸双氧铀(EMS,Ç atalog号码:22400)
甲醇(得自Mallinckrodt,Ç atalog号:3016)
聚/床812嵌入试剂盒用DMP-30(自Polysciences,Ç atalog号:08 792和08791 )
干冰(内部供应)
双去离子水(内部供应)
干式液氮(LN 2 )(内部供应)
十二碳烯基琥珀酸酐(DDSA)(Polysciences,目录号:08792,试剂盒与35相同)
纳迪克(NMA )(Polysciences,目录号:08792,试剂盒与35相同)
改良的Youngren's,只有BACTO-蛋白p(MYOB)板用于蠕虫维护(请参阅食谱)
胆固醇储备溶液(请参阅食谱)
大肠杆菌OP50库存(请参阅食谱)
纤维素毛细管附件(请参阅食谱)
20%BSA解决方案(请参阅食谱)
25 mM Levamisole解决方案(请参阅食谱)
快速冻结替代(QFS)鸡尾酒(请参阅食谱)
聚/ B ed 812树脂混合物(请参阅配方)


设备


微量移液器(任何品牌,容量分别为1 µl,20 µl和1,000 µl)
尖头镊子(EMS,Dumont,目录号:78320-51T,72919-0A和78340-51S)(图1a-1c)
用PVC绝缘的镊子(EMS,Dumont,目录号:3C-linox-E)(图1d)
用PVC绝缘的长钳(Leica,VOMM,目录号:22SAESD)(图1e)
压线钳,解剖刀#20(Bard-Parker,目录号:371620)(图1f)




图1.样品处理工具。交流 用于样品处理尖锐点镊子(第甲小号TEP š 4-5)。德 绝缘镊子冷冻样本恢复和传递(部分B小号TEP小号4-9,和C部分小号TEP小号6-7 )。F。压接工具:#20手术刀通过在金属表面上轻轻摩擦刀片来手动钝化(McDonald等,2010)。


顶板centrifu GE仪(Eppendorf,型号:5424或类似的)
带磨砂玻璃台架的底部照明立体显微镜(Leica ,型号:SM2745或类似产品)
徕卡高压冷冻机,超低温配备有体视显微镜和漏斗填充LN 2 (莱卡,米Odel等:ICE)
冷冻样品回收冷冻箱(不锈钢托盘,深),带有冷冻样品释放站和3毫米冲头,杆和塞子(奥地利莱卡; EM ICE高压冷冻机包装)
迷你台式轨道振动器(VWR,目录号:97109-890)
大型表面滑动加热器(高级,目录号:XH-2002)
红外测温仪(通用工具和仪器,目录号:IRT207)
分析天平(Sartorious Ç ORP,米Odel等:BCE64-15)
搅拌板(Corning ,目录号:PC420D或类似产品)
连接到机械泵标准干燥器(特德佩拉,米Odel等:VRD4)
带12毫米孔的金属块(ThermoFisher Scientific,目录号:88880152)
干冰桶,多个(VWR,Scienceware Magic Touch 2带盖,货号:M16807-2001)
本生灯
化学安全罩
实验室计时器
化学规模(梅特勒,米Odel等:AE240)
温度控制烘箱(昆西实验室,米Odel等:20GC)
标准高压釜
吹风机(任何带热风风扇的品牌)
大型LN 2杜瓦瓶样品存储(泰勒-沃顿,目录号:HC34)
25升滚动底座上的便携式LN 2杜瓦瓶(沃辛顿,目录号:LD25)
P ortable LN 2杜瓦瓶,4 L(沃辛顿,目录号:LD4)
用于处理LN 2 (任何品牌)的PPE(实验室外套,面罩和耐寒手套)
外科口罩(船上卫生,货号:28806;或任何品牌)


程序


秀丽隐杆线虫的单个胚胎的高压冷冻(HPF)
通过将20-30饥饿L1开始蠕虫培养幼虫上的新MYOB琼脂平板种子ED与大肠杆菌OP50细菌(见ř ecipe小号)。每隔第四天早上将2-3只成虫转移到一个新的MYOB盘中以保持蠕虫的生长(Stiernagle,2006年)。
在进行HPF实验前72小时,将5-6例怀孕的成年人转移到几个新的MYOB平板中(至少3个单独的平板以确保有足够的年轻成年人)。
在HPF实验的早晨,取出的20%w / v的BSA溶液两等份(500微升各)从4℃和自旋在94 ×g下(〜1000转)为5分钟在台式离心机以除去气泡。保持在室温下。
在解剖镜旁边放置3-4对干净的尖头镊子(图1a和1b),在Leica ICE高压冷冻机旁边放置1-2对(图1c)。镊子1a和1b适用于纤维素管的转移,而镊子1c适用于木板的转移。它是有旁镊子(图2个酒精棉临界一个):除非你清理它们频繁,镊子变粘用20%BSA溶液,转移期间导致样品损失。
代替一个微量(1微升)设定为0.8微升和另一微量(20微升)定为12微升旁边醇拭子,镊子,卷边工具(白色箭头),和蠕虫拾取(黄色箭头),如图2一个。设置蠕虫解剖工具(带有21G针头的注射器,图2b )。将多个纤维素管连接到移液器吸头上(图2c ,紫色箭头和插图)。保留一根专用于毛细管处理的微量移液器(1 µl)(图2c )。
取出35毫米9-10片状物(A型和B型分别)P ETRI菜(图2 Ç )中,用黑色标记标记A型腔,将其从B型后区分。
注:Ť YPE阿片状物具有在一个面上的单个300μm的深腔,而另一面是平坦的; 样品将在后面B型扶乩(的浅腔被放置小号TEP甲14 )。




图2. HPF之前的蠕虫解剖设置。一种。微量移液器(1 µl和20 µl),尖头镊子,酒精拭子,压接工具(白色箭头)和蠕虫镐(黄色箭头)。b。蠕虫解剖工具–在2.5 ml注射器上使用一对21G针头,用作剪刀来剪断蠕虫。C。专用的微量移液器(1 µl),用于处理与移液器吸头相连的纤维素毛细管(紫色箭头和d)。币坯类型A和类型B被放置在单独的塑料P ETRI菜肴。d。甲纤维素毛细管附件(见ř ecipe 4)胚胎和/或蠕虫集合。


检查液氮(LN 2 )运输杜瓦小号,以确保它们是空的和干燥。用LN 2填充一个大杜瓦瓶(25 L)和两个中等杜瓦瓶(4 L)。
打开Leica ICE高压冷冻机(图3a),等待听到压缩机打开(由嗡嗡声和轻微的振动确定),这也应反映在监视器状态屏幕中。装载站(图3b)应清洁干燥。用25 L杜瓦瓶中的LN 2向冷冻室(图3c)缓慢填充,并进行多次短暂的停顿,以避免触发警报和错误指示储罐已满。在冷却水箱的过程中,大量的LN 2蒸发。
注意:您可能需要第二人的帮助才能安全地倒入〜18升LN 2 。

组装HPF样品存储杜瓦瓶(图s 3d- 3 f)。请注意,杜瓦瓶中的三分容器可以在三个杯子中的每个杯子中最多容纳三个滤芯系统,这相当于在样品存储杜瓦瓶装满之前最多可以进行9次HPF“注入”。高压冰柜需要约20分钟才能达到LN 2温度。一旦平衡,填充HPF样本存储杜瓦(图3F)与LN 2和插入件插入徕卡ICE冷冻室(图3g,在装载站下方˚F igure 3A)。您必须等到冷冻室准备就绪为止,否则容器将收集并冻结空气中形成冰晶的冷凝物。与其他冷冻实验一样,在此过程中必须避免结冰或结霜。因此,请保持干燥条件,并迅速进行工作,以最大程度地减少样品和工具在潮湿环境中的暴露。
组装(图在Leica ICE高压冷冻装载站的两半塑料适配器小号3H- 3在图3b中j和箭头)。将带有小孔的刮板支架(图3i)放在底部钢表面上的塑料适配器顶部(图3b,黄色箭头)。手动关闭红色盖子(图3b),以确保冷冻机正常工作(图5),以运行空白的HPF循环。






图3.为HPF设置Leica EM ICE。一种。徕卡EM ICE高压冷冻机。b。装货站,从3a中的方框区域放大。箭头显示了每次HPF运行时放置板托的位置,顶部有一个半圆柱体(白色箭头和3h),在带有另一个孔的板托(3i)之上,另一个半圆柱体位于底部黑色钢制半块(黄色)上。箭头和3j)。C。LN 2加气口在灰色门后(黑色长箭头)。df。HPF样品存储杜瓦瓶的组装,包括三分体荚(3d)和分段插件(3e中的灰色圆柱体)。3e中的插图显示了组装后的俯视图;b箭头,“刺刀”或释放按钮和黄色箭头,锁定/打开位置。卡口被按压并旋转计数器-clockwise或顺时针以打开或锁定分别(左插图-刺刀一吨打开位置和右插图-刺刀在接近的位置)。G。收集支架单元在自动撤回冷冻室单元之前已插入容器中,在3a中的装载工位下方显示为黑色边框。带有“ in>”标签的凹口(插图3e ,黄色环)应面向HPF仪器固定装置。hj。徕卡墨盒系统包含两个相同的半圆筒和一个带有孔的平板支架。


用蠕虫采摘收集3-4个年轻的成年蠕虫,并将其滴入(12 µl)20%BSA溶液中,放在干净的载玻片上。用两根针作为剪子(图4a)将蠕虫切开(在身体的中部)以释放胚胎(McDonald,1999; Muller-Reichert等,2007 ;McDonald等,201 0 )。
注意:如果收集到完整的蠕虫,请使用含25 mM左旋咪唑的20%BSA溶液并孵育2-3分钟以麻醉蠕虫。可以将蠕虫单独吸收到毛细血管中,如下所示。

目视扫描胚胎,以找到处于胚胎发育所需阶段的胚胎,并牢记导致HPF的步骤大约需要2分钟。避免在解剖范围内长时间观察,因为少量20%BSA溶液会因蒸发而迅速变干。
收集期望的级的胚胎,放置一个纤维素毛细管的开口端,安装于移液管尖端(图4b ,卡通)接近胚胎。由于毛细作用,胚胎将进入毛细管,因此无需将其移液(图4c,卡通)。
使用压接工具,以在毛细管轻轻按下,(否则会切开胚胎就会飘走)上被困胚胎两侧密封毛细管(参见图4c,卡通)。我们Ë相同的工具来与被困胚胎的部分(图4c,插图)从解剖蠕虫中分离掉。请记住,该部分的长度应保持在2 mm左右或以下(图4c,插图),以使其适合B型平舱的空腔(Muller-Reichert等,2008)。按照胚胎发育的胚胎被困的下一个解剖范围。
在所需的胚胎发育阶段前约1分钟,将0.8 µl的20%BSA溶液添加到100 µm的B型小板腔中(图4d)。必须将刮板填充到顶部,以免产生气泡,但不能过度填充,以防止过多的液体芯吸到装载区域。柔和的正弯月面通常就足够了。用尖头镊子将带有胚胎的毛细管部分(图4c,插图)转移到装有20%BSA溶液的B型小板中(为便于观察,图4d中显示了带有完整蠕虫的毛细管部分)。将A型预检板(平侧)放在B型预检板的顶部(图4g)。
转移的扶乩小号andwitch与胚胎固定内部(图小号4E-G ),以的徕卡EM ICE装载站(图3d中,预组装cartridege系统;还参见˚F igure小号4H-ⅰ)。手动关闭红色翼片(图4J)将启动冷冻过程,其中所述盒系统内固定的胚胎被冷却到LN 2项在几十毫秒内和之下的约2高的压力下,000巴(参照图5) 。


图4. HPF的样品制备。交流 在20%的BSA溶液中解剖幼虫。在每个图像上方的卡通图中描绘了步骤。将选择的胚胎收集到纤维素毛细管中,并用压接工具(c,插图)捕获。d。Planchette,在20%BSA溶液的纤维素毛细管内部充满整个蠕虫,并充满顶部。例如。上的锐点镊子,其用于将毛细管片(C传输,插图),以所述B型扶乩空腔,用于放置的A型扶乩(平坦侧)之上的B型扶乩。hj。将刨板三明治(g,右)牢固地放置在支架中(h,橙色箭头)。中号全线关闭红色盖(j)的装载站信息将样本投入到LN 2未DER高压。




图5.高压冷冻循环的温度和压力图。x轴是时间(以毫秒(ms)为单位),y轴是压力(以巴为单位,红色)或温度以开尔文(K)为单位,蓝色。仅出于说明目的,这是从高压冷冻机一次运行的可接受执行的屏幕截图,在样品冷却以18163 K / s的速率(dT / dt)开始时,压力读数为2237 bar。使用缩小按钮(右下角)压力,可以获得长达600毫秒的温度状态(请参见插图)。有关详细信息,请参阅Muller-Reichert等。(2007年和2008年)。


重复小号TEPS一个10一个15 ,直到你冷冻样品的必要数量。在Leica ICE高压冷冻机中,单个样品存储杜瓦瓶的最大容量为9个样品,但是通常在实验开始时执行空白HPF运行,最多可容纳8个样品。需要注意的是它可以将样品收集切换到新的和干燥的支架(图3D),以收集另一组向上的九个样品。其他高压冷冻机没有9个样品的限制。
高压冷冻样品可以在LN 2下保存多年。要长期保存样品,请转到协议B。要进行冷冻替代,请直接转到协议C,而跳过协议B。


从HPF机中回收冷冻样品
要存储冷冻样品,请用一把锋利的迷你螺丝刀的热端在侧壁上打孔,准备一个50毫升的聚丙烯管(将尖端短暂地放在本生灯上加热)。该孔可防止压力升高,并在意外变暖的情况下防止爆炸的危险。将长(约3英尺)长的绳子牢固地固定在盖子上(图6),并贴上胶带标签,以便将装有冷冻样品的试管从大型LN 2储罐中拉出。




图6.储存管的准备。如在部分B所述制备的50ml蓝帽管小号:TEP 1.插图甲管和磁带标签到串上N实施例的标签。


通过在其侧壁上打孔准备多个1 ml冷冻管(类似于S tep B 1)。该孔可防止管道中的压力积聚。用铅笔在每个冷冻管上标记日期,实验编号和样品编号。铅笔标记是丙酮和帮助稳定防止混合-起来之后。
使用吹风机将样品卸载腔室和附件单元中的水分完全清除(图7a)。用LN 2缓慢填充腔室,然后等待直到没有气泡为止,即LN 2表面是平静的。保持有机玻璃盖处于关闭状态,以最大程度地减少冰或霜的污染。
将所有镊子和其他配件放在温度为45°C的隔间附近的滑动加热装置中,并保持电吹风机的插入状态,以备再次使用时使用。快速并交替使用镊子,将镊子放在滑动加热器上,以防止冰晶沉积到样品卸载室中。
注意:戴外科口罩可减少充满LN 2的腔室中的凝结和雾气。

从Leica ICE高压冰箱中取出杜瓦瓶样品。将分割插入物和连接的三等分荚(图7c)转移到冷冻箱中的指定插槽(图7a,左侧),并通过按压和扭转“刺刀”来释放包含切板组件的三等分荚(图s 7c- 7d )。 ”,或从分段插入件上的锁定到打开位置释放按钮(图7c ,蓝色箭头)。
将干燥的绝缘镊子(图1d)浸入LN 2中,使其远离样品约20 s,以将其冷却至LN 2温度。手表中的气泡,并等到了嘶嘶声停止小号。
一次从样品架中取出一个塑料适配器(图7e),仅在支架上留有冷冻的小刀。Q uickly保持器与冷冻片状物传送(在LN保持浸没2所有的时间)到所述样品释放站(图小号7E -7F ,右侧)。
用释放手柄(图7a,白色箭头),将冷冻的小刀从刀柄中冲出到容器中(图7g,黄色箭头)。在不传送/释放样品时,保持有机玻璃盖处于关闭状态,以最大程度地减少冰污染。
将预先标记的冷冻管放入冷冻箱中,并冷却至LN 2温度几分钟。我们ë干镊子(图1D)至TR ansfer冷冻币坯成各自的冷冻管(在LN保持浸没2的所有时间)。用您的手掌预热每个冷冻管帽,然后迅速合上。如果瓶盖不保暖且无水分,则它将冻结在试管中,并且几乎不可能在不预热整个试管的情况下重新打开试管,从而损坏冷冻样品。




图7.高压冷冻后的样品回收率。为了清楚起见,所有照片均在没有LN 2的情况下拍摄。一种。样品回收冷冻箱显示了一个用于样品架的托盘(左)和一个带有释放手柄的样品释放站(白色箭头),右侧是收集容器。bd。ICE高压冰箱的样品架将红色转移到样品回收冷冻箱中。逆时针旋转bayon等(c,蓝色箭头)可将样品收集容器单元从气瓶中释放出来。例如。一次将一个样品架转移到释放站。冷冻的小球被收集到水库中(g,橙色箭头)。


最终检查:检查是否有裂开的小方舟,如果发现,请收集没有黑点的小方舟。用作封面的A型预检器(平底)上标有黑点,并且由于冷冻胚胎在未标记的B型预检器的腔内,因此很容易在LN 2下识别并丢弃。
将所有冷冻管收集到预先贴有标签的50 ml聚丙烯管中(在便携式杜瓦瓶中在LN 2中进行预冷),用盖子盖上盖子,并在外面看到带标签的细绳(请参见图6),然后将其放入LN 2储罐中。样品可以适当地保存多年。


冷冻替代HPF胚胎用于电子显微镜
注意:此过程总共需要3-4天,由于孵育时间长,第一天可能需要8小时以上。确认您有足够的干冰和LN 2供应。试剂必须是新鲜准备的,因此请相应地计划您的一天。使用滑动加热器和吹风机使所有乐器保持无水分!

1.按照配方7 (Rahman等人,2020年)准备QFS鸡尾酒,并在预先标记的冷冻瓶中各装1毫升等分液(按样品编号标记,每小瓶一个冷冻样品)。     

2.将一个带有12毫米孔的金属块朝上放在干冰桶中。填充LN 2并冷却至LN 2温度。经常添加LN 2以保持该块被淹没。在桶上盖上盖子,以防止结露。桶必须放置在化学通风橱中,随后的步骤会在通风橱中进行,因为是有毒且易挥发的。     

3.通过将试管放入浸没在LN 2中的金属块中来冷冻QFS混合物。不要添加过多的LN 2 ,否则管子会从块中浮出。     

4.从LN 2储罐中收集样品冷冻管(HPF胚胎或蠕虫)到装有LN 2的杜瓦瓶中(图8a,蓝色箭头)。     





图8. QFS的样本传输。一种。冷冻样品在LN 2杜瓦瓶中转移(底部,蓝色箭头)。QFS鸡尾酒浸入LN 2的干冰桶中(左上方,黄色箭头)。b。将装有冷冻QFS鸡尾酒冷冻管(左上)和HPF样本冷冻管(右上)的浅聚苯乙烯泡沫塑料容器平放在LN 2中。一次打开一个试管,打开冷冻管(右上),将冷冻的小球倒入浅聚苯乙烯泡沫塑料容器中的LN 2中,然后转移到与i n S放在同一平面上的冷冻管中的冷冻QFS混合物中(左上)。TEP小号C5到C7用镊子。C。将QFS管横向放置在干冰上(在预冷的金属块中)。d 。桶中装满了更多的干冰,盖上盖子,然后放在轨道振动器上(McDonald和Webb,2011年)。


5.取一个干净干燥的浅泡沫聚苯乙烯泡沫塑料容器或托盘。通常15 × 20厘米深2厘米的运输容器盖就足够了。在托盘上添加LN 2并使其冷却,然后再进行后续步骤。     

6.一次取出一根冷冻管。打开冷冻管的盖子,并迅速将小瓶平放在装有LN 2的聚苯乙烯泡沫塑料浅容器中,并从LN 2下方的小漏斗中倒出。在它旁边,也要打开瓶盖并在LN 2中放平并放置一个冷冻的装有QFS的样品瓶。将该样品瓶的瓶盖保持在您的手掌或滑动加热器中,以免结露,否则会结冰并被卡住。     

7.用尖锐的镊子将装有冷冻QFS鸡尾酒的小瓶转移到每个小瓶中。快速站起冷冻管(将大部分样品瓶浸入LN 2中),然后将保暖帽拧到试管上。立即将其转移到在LN浸没金属块2 (小号TEP Ç 2 )。如果可以轻松识别样品,则可以在单个QFS鸡尾酒管中放置多个小菜刀,但是我们警告说,小菜刀在QFS期间会相互碰撞并移走大样品。     

8.经常检查并添加LN 2,以使金属块始终浸没在水中。将盖子放在顶部,以尽可能防止空气中的水分凝结。     

9.重复小号TEPS Ç 4到C 7的未直到把所有样品含有币坯被转移到单独标记含有QFS-冷冻管。我们建议一次不要尝试超过八支试管。     

10.小心倾倒LN 2,并在干冰桶的一半处注满冰块。旋转金属块,这样冷冻管现在可以平放在一边。加入更多干冰以完全覆盖金属块,并用盖子盖住桶。将其放在化学安全罩内以60个循环/分钟的速度放置的轨道振动器上放置3小时(McDonald和Webb,2011年)。 

11. 3小时后丢弃干冰。放回盖子,并继续以60转/分钟的速度旋转一个小时。 

12.取下盖子,并继续以60转/分钟的速度摇动另外一个小时(或根据需要),直到金属块达到约4°C的温度为止。经常用红外测温仪检查温度。这是关键的一步!在4°C或更高的温度下长时间暴露可能会使整个体积显着变暗,从而难以识别毛细管内的单个胚胎。 

13.用100%丙酮(储存在4°C下)代替QFS混合物。首先,用玻璃移液器小心地移出QFS鸡尾酒(注意不要让Planchette受到干扰)。使用新鲜的移液器,将溶液(100%丙酮)释放到冷冻管的侧壁上,轻轻地重新注满。每次孵育1小时后,用以下混合物替换溶液(使用定为60个循环/分钟的定轨振荡器)。 

1:2树脂:丙酮
1:1树脂:丙酮
2:1树脂:丙酮
根据机构生物危害处置程序,丢弃过程中使用的所有移液器,用过的和未使用的试剂,纸巾和Kimwipes。

14.将浮游植物转移到100%的树脂中,并在室温下在设定为60个循环/分钟的定轨振荡器上放置过夜(14h至16h)。 

15.第二天早晨,用新鲜准备的100%树脂代替隔夜树脂,然后立即进行下一步。 

16.用剃刀刀片切开Beem胶囊的底部;打开胶囊的盖子并弯曲铰链,使盖子平放在表面上。将刮板样品一面朝上,并在Beem胶囊盖的内部放几滴树脂。小心地将胶囊封闭在盖子上,使切板样品的一面朝上。带有切口孔的胶囊底部应朝上。通过此开口用100%树脂填充胶囊(请参见图s 9a- 9 i),然后将其烘烤以在75°C的烤箱中固化60 h至65 h。 

17.一旦固化,用剃刀切断塑料光束胶囊(参见图小号9j- 9米)。现在,在解剖范围内,小心地去除金属平板周围的所有树脂。重要提示:请确保完全除去侧面的所有树脂-切板侧面的发亮金属应完全暴露(请参见图s 9o -9 p)。现在,快速将Planchette的裸露金属基底浸入LN 2中,直至达到低温,然后用吹风机加热,直到所有冷凝物消失并且Planchette变热。根据需要多次重复冷/热循环,直到由于金属和树脂之间的热膨胀差异而导致切板或金属载体弹出并掉落。这将使树脂包埋的样品完好无损(图9r)。不要用武力松撬扶乩,因为这种风险小号压裂树脂而使树脂包埋样品仍处于腔。在切片进行成像(TEM或vEM)切片之前,您可以在室温下在防尘存储容器中放置有或没有附有刮板的树脂嵌入样品多年。 



注意:此处描述的QFS协议足以用于我们手中的FIB-SEM成像和阵列层析成像。对于常规的TEM成像,可以将样品切开并通常进行后染色以增强对比度(Hayat,2000)。对于其他体积EM方法,例如串行块面SEM,用户可能希望通过适应室温整块染色规程(Hayat和Giaquinta,1970; Hua et al。,2015)来进一步增强样品的金属化能力,以进行高分辨率工作。。




图9.树脂嵌入和样品处理。交流 用剃刀刀片切开Beem胶囊的底部。德 弯曲铰链,使盖子平放在表面上。放置扶乩(从QFS小号TEP Ç 15 )样品的一面朝上,并添加树脂几滴用巴斯德吸管。fg。小心地完全合上盖子上的胶囊,保持平板的样品面朝上。你好。用100%树脂填充Beem胶囊,然后转移到烤箱中(请参阅S Cep C 16)进行固化。固化后,将染色的样品是在所述胶囊(9底部Ĵ ,后视图)。K-米。使用剃须刀卸下Beem胶囊。np。ü的nDer解剖范围使用小心除去树脂剃刀刀片以暴露扶乩。q。一旦扶乩暴露多次,沉浸在它LN 2 ,接着加热以一个吹风机,直到金属载体弹出。河 去除木板或金属载体后,将暴露的样品包埋在100%的树脂中。


笔记


使用液氮可能存在危险。必须使用适当的个人防护设备(PPE)保护您的眼睛,面部和裸露的皮肤。另请参阅您所在机构的安全程序。

s是重金属,如果吸入会危害健康。你必须戴上口罩,并准备将在连接到排气系统的化学罩冷冻替代鸡尾酒组合。


菜谱


改良的Youngren's,仅BACTO-蛋白p(MYOB)板用于蠕虫维护
Bacto琼脂20克                         

氯化钠(NaCl )2克                                         

盐酸Trizma 0.55克                         

Trizma-OH 0.24克                         

Bacto P蛋白one 3.1克                         

去离子水至1升

高压灭菌20分钟(液体循环),使其冷却
甲DD1.6毫升胆固醇从库存soluti上(见下文)
中号有磁力搅拌器之前彻底IX至倒入35mm组织培养皿中,大约每盘5 ml(约每升200个板)
琼脂平板凝固并干燥后,应用约200 µl大肠杆菌OP50培养物(见下文),在室温下静置两天,直到细菌溶液干燥并形成细菌草皮为止(Church等, 1995)
胆固醇储备液
胆固醇5毫克                         

乙醇(200标准)100毫升           

使用小型磁力搅拌棒溶解胆固醇(慢速约2小时)

大肠杆菌OP50库存
从冷冻原液中,在琼脂平板上划线细菌(不使用任何抗生素),并在37°C下孵育过夜(〜14-16 h)。将少量分离的菌落接种到100 ml LB培养基(不含任何抗生素)中,并在37°C孵育6-7小时,不要摇晃。种子MYOB板用200μl的培养(Stiernagle,2006) 。适当处置未使用的细菌培养物。

纤维素毛细管附着
参见图10




图10.用于样品收集的纤维素毛细管附件。用锋利的剪刀将纤维素毛细管切成约4厘米的碎片。下一个解剖范围,推动或拉动穿过1个微升移液管尖端(无毛细一个屏障),直到毛细管的边缘接近尖的边缘。适用指甲油粘上毛细管的移液管尖端的前端,并允许它在室温下为一小时干燥(穆勒-赖克特等人,2003) 。确保毛细管的另一端打开。如果不是,请用锋利的剪刀(s箭头)将其切开至边缘附近。


20%BSA解决方案
              牛血清白蛋白10克                         

              M9缓冲液至50毫升           

准备在多个步骤中的溶液,即,各添E添加少量BSA的成35毫升的M9缓冲液(在50毫升蓝帽管)
中号轻轻IX上的桌子顶部轨道摇床在低速,以避免产生气泡
一旦BSA完全溶解,使其体积达到50 ml 。轻轻混合至均质,并以小等分试样在4 ° C或-20 ° C下保存(长期保存),最多可保存一年
25 mM左旋咪唑溶液
在10 ml的20%BSA溶液中添加0.051 g左旋咪唑
在台式轨道振动器上低速缓慢混合,以免产生气泡
注:钍é解决方案可用于高达两到三个月。

快速冻结替代(QFS)鸡尾酒
注意:必须准备一个化学防护罩。使用硼硅酸盐玻璃移液器测量丙酮和甲醇。请跟随机构人处理锇和铀化合物化学品安全管理条例。这些都是剧毒的。安全至上。

OsO4颗粒0.1克           

丙酮13.5毫升           

在室温下搅拌至完全混合约10-15分钟。一旦看不到残留物,则从新打开的小瓶中添加:

0.75毫升甲醇中的2%乙酸铀酰(UA)                         

蒸馏去离子水(DD2)0.75毫升           

在室温下完全混合并通过0.22 μ醋酸米纤维素过滤器,以除去任何残留的未溶解的元素。甲liquot1毫升成单独的冷冻管中,在LN闪光冷冻2和储存在- 80℃ (拉赫曼。等人,2020)

聚/ B ed 812树脂混合物
聚/ B ed 812 14.6克             

十二烯基琥珀酸酐(DDSA )8.4克                           

纳迪克甲酸酐(NMA )7.0克                           

DMP-30 0.42毫升           

用磁力搅拌器轻轻混合直至均匀。从试剂盒中准备新鲜食物,其中包含上述所有化学物质(Rahman等,2020)


致谢


我们感谢希瑟·贝伦斯曼(Heather Berensmann)对手稿的认真阅读和摄影方面的帮助。

MM Rahman和O. Cohen-Fix得到了美国糖尿病与消化与肾脏病研究所的资助(校际资助DK069012)。IY Chang和K. Narayan得到了美国国家癌症研究所(合同号75N91019D00024 )的支持。根据合同号75N91019D00024,该项目已全部或部分由美国国立卫生研究院国家癌症研究所的联邦资金资助。本出版物的内容不一定反映卫生和公共服务部的观点或政策,也没有提及商品名称,商业产品或组织,这暗示着美国政府的认可。


利益争夺


作者宣称没有任何竞争性的经济利益。


伦理


危险化学品是根据机构设置人的指导方针。


参考


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马萨诸塞州哈亚特(2000)。电子显微镜的原理和技术:生物学应用(第4版)。英国剑桥;纽约:剑桥大学出版社。
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McDonald,K。(1999)。高压冷冻保存高分辨率的精细结构和免疫标记的抗原性。在:Hajibagheri,N.(Ed。)。分子生物学中的方法:电子显微镜方法和规程(第117卷,第77-97页)。新泽西州托托瓦:Humana Press Inc.
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McDonald,K.,Schwarz,H.,Muller-Reichert,T.,Webb,R.,Buser,C. and Morphew,M.(2010)。用于模型系统高压冻结的“技巧”。方法细胞生物学96 :671-693。
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Copyright: © 2021 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. Rahman, M. M., Chang, I. Y., Cohen-Fix, O. and Narayan, K. (2021). A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy. Bio-protocol 11(7): e3981. DOI: 10.21769/BioProtoc.3981.
  2. Rahman, M., Chang, I. Y., Harned, A., Maheshwari, R., Amoateng, K., Narayan, K. and Cohen-Fix, O. (2020). C. elegans pronuclei fuse after fertilization through a novel membrane structure. J Cell Biol 219(2). doi:10.1083/jcb.201909137.
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