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Jan 2017

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Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus
GFAP和TNF-α在小鼠下丘脑的免疫荧光   

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Abstract

Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.

Keywords: Hypothalamus (下丘脑), Immunofluorescence (免疫荧光), Cryosections (冰冻切片), Neuropeptides (神经肽), Glial Fibrillary Acidic Protein (GFAP) (胶质纤维酸性蛋白(GFAP)), TNF-α (TNF-α), Tumor necrosis factor alpha (肿瘤坏死因子-α)

Background

Elucidating the protein organization in neurons and glia is essential for understanding hypothalamic function. Immunofluorescence is a powerful tool to investigate changes in the expression of neuronal and glial proteins and to map activated brain areas at the cellular level. This imaging technique enables the visualization of highly specific protein (antigen) targets using a combination of primary and secondary antibodies. Antigenic determinants of interest in brain tissue are first treated with a monoclonal or polyclonal primary antibody, targeted toward peptides from the species/protein of interest generated in a host (mouse-, rabbit-, chicken-, rat-, donkey-derived, etc., are available), followed by a secondary antibody directed toward the host of the primary antibody and conjugated to a synthetic or natural fluorophore. The antibody targets often labeled in brain tissue sections are glial fibrillary acidic protein (GFAP), neuronal and axonal neurofilaments, neuropeptides, microtubule-associated proteins, blood-brain barrier proteins, pre- and post-synaptic proteins, myelin, markers of inflammation, and numerous antigens associated with specific diseases. Among the useful fluorescent markers for the visualization of antigens in brain tissue are rhodamine, fluorescein, the Alexa Fluor series, and the cyanine dyes. Counterstaining for nuclei using a variety of popular DNA-binding dyes (such as TO-PRO-3 iodide) follows treatment with the secondary antibody.


This method has been previously used by our laboratory to identify neuronal subpopulations in various hypothalamic nuclei involved in energy homeostasis (Dalvi et al., 2017). In particular, using double immunofluorescence staining, we identified GFAP in the hypothalamus and tumor necrosis factor (TNF)-α, a marker of inflammation, in the hypothalamic arcuate nucleus following acute lipid infusion and chronic high-fat diet feeding in mice, which may contribute to a significant alteration in content or expression of appetite-regulating neuropeptides. To the best of our knowledge, this was the first description of TNF-α induction demonstrated by immunofluorescence in the mouse hypothalamic arcuate nucleus after exposure to a high-fat diet. This method can be applied to the identification of other markers of inflammation and endoplasmic reticulum (ER) stress in the hypothalamic regions provided that reliable antibodies are available for detection. This protocol details a generalized procedure for staining frozen brain cryosections ranging from 20 to 30 µm in thickness. Figures 1 and 2 presented in this protocol are confocal images that reveal the expression of GFAP and TNF-α in the hypothalamic arcuate nucleus in 25 µM brain sections of mice treated acutely with lipid or chronically with a high-fat diet (Dalvi et al., 2017).



Figure 1. Effect of acute intralipid treatment at 24 h post-infusion in C57BL/6 mice on hypothalamic arcuate nucleus (ARC) reactive astrocytosis and expression of TNF-α, a marker of inflammation. A-B.Representative images showing immunofluorescence for the expression of GFAP (green) and TNF-α (red) in the hypothalamic ARC of C57BL6 mice at 24 h following acute infusion with saline (A) or Intralipid (B). C. Quantification of the immunofluorescence intensity for GFAP and TNF-α expression in the ARC of C57BL/6 mice at 24 h following acute infusion with saline or Intralipid. Data in the bar graph are expressed as the mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control. Images in A and B and the graph in C are adapted and modified from Dalvi et al. (2017).



Figure 2. Effect of high-fat diet (HFD, 60% kcal) feeding for 8 weeks in CD-1 mice on reactive astrocytosis and expression of TNF-α in the hypothalamic arcuate nucleus (ARC). A-B. Representative images showing immunofluorescence for the expression of GFAP (green) and TNF-α (red) in the hypothalamic ARC of CD-1 mice fed chow (A) or an HFD (B) for 8 weeks. C. Quantification of immunofluorescence intensity of GFAP and TNF-α expression in the ARC of CD-1 mice fed either chow or an HFD for 8 weeks. Data in the bar graph are expressed as the mean ± SEM (n = 5-6 animals/group); *P < 0.05 versus control. Images in A and B and the graph in C are adapted and modified from Dalvi et al. (2017).

Materials and Reagents

  1. 50 ml Falcon tubes

  2. Aluminum foil

  3. Fine paint brushes

  4. Cryostat blades (Leica Biosystems, catalog number: 3802106-TRI-FACET/LOPRO/CTD Blade-10PK)

  5. 12-well plates to store frozen sections (BD Falcon, Corning, catalog number: 353043)

  6. 24-well plates to store frozen sections (BD Falcon, Corning, catalog number: 353047)

  7. Coverslips (25 × 50 mm) (VWR International, catalog number: 48393-059)

  8. SuperFrost Plus slides (positively charged) (Thermo Fisher Scientific, catalog number: 12-550-15)

  9. Wax pen (PAP Pen) (Abcam, catalog number: ab2601)

  10. Fresh 4% paraformaldehyde in PBS (75 ml per mouse) (Millipore Sigma, catalog number: 158127-100G)

  11. 0.9% saline (Millipore Sigma, catalog number: S8776-100ML)

  12. PBS pH 7.4 (Thermo Fisher Scientific, Gibco, catalog number: 10010023)

  13. 15% and 30% sucrose in PBS (300 g sucrose in 1 L PBS can be stored in the fridge for up to a month) (Millipore Sigma, catalog number: S7903-1KG)

  14. Cryoprotectant solution (Millipore Sigma, catalog number: G7893-500M)

  15. Tissue embedding solution (Tissue-Tek® O.C.T. compound) (Sakura® Finetek, VWR, catalog number: 25608-930)

  16. Tween 20 for preparation of PBS-T (0.06% Tween-20 in PBS) (Millipore Sigma, catalog number: P1379-500ML)

  17. Triton X-100 for preparation of PBS-Triton-X100 (0.4% Triton-X100 in PBS) (Millipore Sigma, catalog number: X100-500ML)

  18. Normal donkey serum for preparation of blocking buffer (5% serum in PBS-Triton-X100) (Abcam, catalog number: ab7475)

  19. Normal donkey serum for preparation of antibody buffer (2% serum in PBS-Triton-X100) (Abcam, catalog number: ab7475)

  20. Nuclear stain: TOPRO (TO-PRO-3 iodide 642/661) (Life Technologies, catalog number: T3605)

  21. Pro-Long Gold anti-fade mountant (mounting medium) (Thermo Fisher Scientific, catalog number: P10144)

  22. Clear nail polish (or equivalent for sealing coverslips)

  23. Primary antibodies:

    1. Anti-GFAP: Chicken-derived polyclonal mouse antibody against GFAP (1:500 dilution) (Abcam, catalog number: ab4674)

    2. Anti-TNF-α: Goat-derived polyclonal mouse antibody against TNF-α (1:50 dilution) (Santa Cruz, catalog number: SC-1350)

  24. Secondary antibodies (fluorescently tagged secondary antibody against species from which primary antibodies are obtained):

    1. Donkey anti-Chicken IgY (H+L) Secondary Antibody, FITC (will interact with the GFAP chicken-derived anti-mouse primary antibody) (Thermo Fisher Scientific, Invitrogen, catalog number: SA1-72000)

    2. Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (will interact with the TNF-α goat-derived anti-mouse primary antibody) (Thermo Fisher Scientific, Invitrogen, catalog number: A-11057)

Equipment

  1. Scalpel

  2. Tweezers

  3. Fine scissors

  4. Spatula

  5. -20°C freezer

  6. Adult Mouse Brain Slicer Matrix (Zivic Instruments, catalog number: BSMAS001-1)

  7. Mouse perfusion apparatus (VWR, Leica Biosystems, catalog number: 10030-382)

  8. Cryostat (Leica, model: CM3050S or similar)

  9. Confocal laser scanning microscope ((Zeiss, model: LSM 510 or similar)

Software

  1. AxioVision 3.0 imaging software (Carl Zeiss GmbH, Jena, Germany)

  2. ImageJ software, National Institutes of Health, Bethesda, MD, USA (Schneider et al., 2012)

  3. GraphPad Prism software, Inc., La Jolla, CA, USA

Procedure

  1. Isolation and preparation of brain tissue for immunofluorescence


    Perfusion of mice and brain extraction
    1. On the morning of the perfusion (or the day before), make fresh 4% paraformaldehyde (PFA) (Gage et al., 2012).

      Note: Prepare this solution either fresh or no more than 72 h in advance and store at 4°C.

    2. Using a transcardiac approach, perfuse with 20 ml cold 0.9% saline (or PBS) followed by 20 ml cold 4% PFA (up to 25 ml maximum over 5-10 min by slow perfusion).

    3. After the mouse is perfused, extract the brain using a scalpel, tweezers, fine scissors, and spatula (Papouin et al., 2018), gently place the brain into a 50-ml Falcon tube filled with 4% PFA (30-40 ml), and shake for at least 6-8 h (but not more) post-perfusion at 4°C.


    Cryopreserving the brains
    1. Prepare 15% and 30% sucrose in PBS (300 g sucrose in 1 L PBS can be stored in the fridge for up to a month).

    2. Place brains in cold 15% sucrose solution in a 50-ml Falcon tube at 4°C on a shaker overnight or until they sink.

    3. Place brains in a cold 30% sucrose solution in a 50-ml Falcon tube at 4°C on a shaker for 2-3 days or until they sink. The brains should then be cut on a cryostat within a week after freezing (as described below).


    Freezing the brains
    1. Once brains have settled to the bottom of the tube, snap-freeze them in an isopentane bath (as described below).

    2. For investigation of the hypothalamus, before freezing the entire brain, cut it coronally anterior and posterior to the hypothalamus, such that the rostral (anterior) cut is just at the optic chiasm and the caudal (posterior) cut is just at the end of the hypothalamus (between the hypothalamus and pons). Use the mouse brain atlas (Paxinos et al., 2001) and a mouse brain slicer matrix to orient the brain anatomy and to accurately cut the brain. The middle cut parts of the brain will contain the hypothalamus.

    3. Get a small container and fill it halfway with dry ice. Add isopentane to cover the ice. Place a small beaker containing some isopentane in the middle of the bath. Then one by one, place the three cut parts from each brain into the beaker to freeze them. They should turn white within 10-15 s.

    4. Once frozen, wrap the brains in aluminum foil, label them, and leave at -80°C until ready to section.


    Sectioning the brains

    Note: Brain sections can be either collected in a 12- or 24-well plate or directly on a glass slide, depending on the region of interest, availability of time, and the cost of antibody. The sections in a 12- or 24-well plates can be stored at -20°C until further use, but sections on a slide need to be processed right after collection. If processing many sections, ideally use a 24-well plate for processing (but this will require more antibody). However, if processing only a few sections, use a slide to use less antibody.


    1. Turn on the cryostat (Leica CM-3050-S) well in advance.

      Note: The machine takes about two hours to cool to -24°C. Make sure this temperature is reached before continuing. Throughout sectioning, ensure that the cabinet temperature remains between -18°C and -24°C. A temperature range may be required, as the optimal temperature for cryostat sectioning depends on the nature of the tissue, whether the tissues have been freshly frozen or pre-fixed with subsequent cryoprotection, the type of cryostat, and how well perfusion worked, etc. If there are problems with cutting, the temperature can be increased/decreased. In particular, if there is a problem with sectioning of pre-fixed brains, try cutting at a lower temperature, as the pre-fixed brains need a very low temperature.

    2. Take the brains to the cryostat on dry ice. If only sections of the hypothalamus are needed, take out only the middle cut part of the brain corresponding to the hypothalamus.

      Note: Work quickly or the brains will deform. Place a pair of forceps on the dry ice with the brains.

    3. Clean the cover and platform of the cryostat with ethanol.

      Note: The lever on the side of the platform needs to be lifted toward you to unlock the platform.

    4. Take out the tissue embedding platforms (O.C.T. molds) from the cryostat with the embedding solution (O.C.T. compound). Cover the top of the platform with the solution, moving in a spiral motion, and place the platform on the dry ice.

    5. Using the cold forceps, quickly transfer the brain to the platform with its caudal end sitting on it and the free rostral end facing upward. Then add more embedding solution around the brain and on top to cover it. Leave these on the dry ice until the solution begins to freeze (will turn white). Place the platform in the holes on the left side of the interior of the cryostat (Figure 3A, 3B).

      Note: Another common method is to pre-freeze tissue in embedding solution on the embedding platform (i.e., O.C.T. compound within O.C.T. mold) before sectioning and to store at -80°C. However, this method has not been tested in our lab, so we are unable to predict the effect of pre-embedding and storage at -80°C on the protein staining.

    6. Once all the brains have been moved into the machine, close the lid and leave it for about an hour so the brains and the cryotome blade reach the same temperature.

    7. Once brains have frozen, position the blade using the black handle at the side of the stage.

      Note: Use extreme caution around the cryostat blades as they are dangerously sharp.

    8. Label the cover and sides of a 24-well plate (include thickness of samples, date, etc.). Fill each well at least halfway with cryoprotectant solution using a larger Pasteur pipette. Use one 24-well plate per each mouse brain.

    9. Lock the embedding platform with sample into the front holder and adjust into position.

      Note: You can move the entire apparatus closer to the blade by pushing the button at the front of the cryostat, rather than just turning the dial.

    10. Ensure the machine is set to the desired thickness using the dial on the cryostat.

      Note: 20-30 µm thickness is often used for immunofluorescence.

    11. Proceed with sectioning the brain (Figure 3C). Use the fine paint brush to transfer sections to the 24-well plate (Figure 3D, 3E).

      Note: You can discard the first few slices as they will likely consist of just mounting medium and not the brain tissue.

    12. Once the brain becomes visible, place the sections in sequential wells within the 24-well plate containing cryoprotectant (Figure 3E, 3F).

      Notes:

      1. Use the mouse brain atlas (Paxinos et al., 2001) to orient the anatomy of the brain (hypothalamic) slices; this needs practice. First, the two lateral ventricles appear at the beginning of the sectioning, and subsequently the third ventricle appears that has hypothalamus on each side.

      2. In adult mouse, the hypothalamus is about 5 mm in length, so there will be about 250 sections with 20 µm thickness and about 166 sections with 30 µm thickness. Thus, if brain sections are collected sequentially in each well of a 24-well plate, each well will contain 6-10 sections from throughout the hypothalamus.

      3. Alternatively, using the atlas, a certain number of brain sections (i.e., about 6-10 sections) can be collected sequentially in each well of a 24-well plate. It must be noted how the sections are collected to avoid confusion at the time of processing for immunofluorescence.

      4. Try to minimize the contact area between the brush and the section when transferring them from the cryostat to the plate to avoid damage. Also, try to lower the section into the center of the well so as to avoid making contact with the sides.

    13. Once complete, cover the plate with plastic wrap and store in a -20°C freezer.

      Note: Be sure to clean the cryostat and embedding platforms after use (most importantly, remove the blade to preserve its lifespan).



      Figure 3. Steps of sectioning a mouse brain and transferring cut sections to cryoprotectant solution in a 24-well plate. A. Tissue embedding platforms (O.C.T. molds) are used, the top of the platforms are covered with the solution moving in a spiral motion, and the platforms are placed on the dry ice. Using cold forceps, the brain is quickly transferred onto the platform with its caudal end sitting on it and the rostral free end facing upward. More embedding solution is added around the brain and on top to cover it sufficiently. B. The platforms with brains frozen on top of them are left on the dry ice until the solution begins to freeze (will turn white). Once all the brains have been moved into the cryostat machine, the lid of the cryostat is closed and the brains are left inside for an hour so that the brains and the cryotome blade reach the same temperature. C. Using the desired thickness on the cryostat (20-30 µM thickness is often used for immunofluorescence), sectioning of the brain tissue is performed. D and E. Using a fine paint brush, the cut frozen brain sections are transferred to a 24-well plate filled with cryoprotectant solution. F. A magnified image of a single well of a 24-well plate filled with cryoprotectant solution and floating brain sections inside.


  2. Labeling of brain sections: Blocking and incubation with primary and secondary antibodies

    1. Take out 24-well plates containing brain sections from the -20°C freezer (each 24-well plate contains brain slices from an individual mouse in cryoprotectant). Take a new 24-well plate and fill all wells with PBS-T (Figure 4A). Transfer brain sections from one or two wells from an individual mouse to one separate well of the new 24-well plate filled with PBS-T (Figure 4B). Use fine paint brushes to transfer the brain sections from the cryoprotectant into PBS-T. The transfer from cryoprotectant to PBS-T will cause the cryoprotectant and embedding medium to diffuse, and the brain sections will spread.

      Note: Prepare at least 2 wells for the negative controls of the primary antibody. This provides more control sections for imaging (the sections can easily get damaged during sequential frequent washing) (Figure 4B).



      Figure 4. Transferring cut sections of brains stored in cryoprotectant solution in a 24-well plate to a new 24-well plate containing 0.06% Tween-20 in PBS (PBS-T). A. A new 24-well plate filled with PBS-T is prepared, and fine paint brushes and containers with clean water for washing paint brushes are kept ready. B. All 24-well plates containing brain sections immersed in cryoprotectant from an individual mouse are taken out, and brain sections from one or two wells from an individual mouse are transferred to a separate well of the new 24-well plate filled with PBS-T. Fine paint brushes are used to transfer the brain sections from the cryoprotectant into PBS-T. At least 2 wells for the negative controls of the primary antibody are prepared using brain sections from 1 to 3 mice.


    2. Wash 4× with PBS-T, 15 minutes per wash on a gyrating platform. For the “wash” steps, each time transfer the brain sections to a new well filled with PBS-T using fine paint brushes (see Video 1). Use separate brushes for the negative control brain sections and start washing with those sections first to avoid any contamination.


      Video 1. Transferring brain sections to a new well using fine paint brush during the “wash” steps.

      Note: Secure the plates properly and monitor the speed – if the speed increases rapidly, the plates may be dislodged if not secured properly.


    3. In between the washes:

      1. Thaw aliquots of serum. The serum should be the same as the host species of your secondary antibody.

        Note: If the host species of both secondary antibodies was donkey, use donkey serum

      2. Prepare blocking buffer using PBS-T.

      3. Prepare extra blocking buffer because it can be diluted further to prepare the primary antibodies.

      4. For blocking the potential background in the tissue, use 1 ml blocking solution per well, and the remaining can be diluted and used to prepare the primary antibodies.

    4. Transfer brain sections into the blocking buffer and incubate for a minimum of 2 h at room temperature or optimally overnight at 4°C.

      Note: 10% serum in PBS-T can also be used to block the tissue but for a minimum of 1 h.

    5. After blocking, transfer the brain sections into the antibody buffer with the appropriate dilution of the primary antibody(s).

      Note: Diluted primary antibodies can be mixed together and incubated simultaneously. Add 0.5 to 1.0 ml antibody buffer to each well in a 24-well plate. Using less antibody will decrease costs.

    6. Incubate for 12-24 h at 4°C.

    7. Wash 4× with PBS-T buffer for 15 min per wash on a gyrating platform at room temperature.

      Note: Start with the primary antibody negative control sections first to avoid contamination with the primary antibodies.

    8. Block the sections with 5% blocking buffer for 1 h to decrease background.

      Note: It is acceptable to skip this step, if necessary, as the tissue should already be sufficiently blocked.

    9. Transfer sections to the antibody buffer with an appropriate dilution of the secondary antibodies and nuclear stain.

      Notes:

      1. Typically, 1:500 dilution is used for FITC secondary antibodies, and 1:1,000 dilution is used for Alexa Fluor secondary antibodies and the nuclear stain (TOPRO).

      2. Use some sections from each group for negative secondary antibody controls. Make a separate 24-well plate with these sections and use a separate fine paint brush to avoid any contamination with the secondary antibodies.

    10. Incubate for 1 h at room temperature or overnight at 4°C.

      Note: Cover with aluminium foil to avoid bleaching of the fluorescent tag.

    11. Wash 4× with PBS-T following secondary antibody incubation on a gyrating platform for 15 min per wash.

      Note: Cover with aluminium foil when possible to avoid bleaching of the fluorophore.

    12. After the final wash, transfer the sections to PBS, and slowly mount the slices onto SuperFrost Plus slides using fine paint brushes.

      Note: Try to minimize exposure of slices to ambient lighting, maintaining the foil cover over the sections when possible. Place the slides inside a slide holder box with a lid to avoid exposing the brain sections to light. The entire procedure of primary and secondary antibody conjugation can be done on a slide. After placing a section on a slide, circle it with wax pen and stain the slides following the above procedure. This will use much less reagents and cut costs substantially with only a few slices.

    13. Wait until all sections on the slide are dry.

    14. Spin the anti-fade mounting media at 1200 rpm for 10 min at 4°C in a centrifuge so that any solid particles will settle at the bottom of the tube and will not create any background signal.

    15. Dispense the minimum amount of anti-fade mounting media and mount a coverslip.

    16. Dry for 24 h at room temperature or 4°C in the dark, and seal edges with clear nail polish.

    17. Capture images from the immunostained sections on a Zeiss LSM 510 confocal upright microscope fitted with a color digital camera and AxioVision 3.0 imaging software. Capture fluorescent images using an identical exposure time without saturating the pixel intensities (Figure 1A, 1B, 2A, 2B).

Data analysis

  1. Use the ImageJ software (Schneider et al., 2012) to measure the intensity of immunofluorescence (densitometry) in a manner such that the samples are not identified to the assessor (blinded).

  2. To measure fluorescence intensity, background is first determined based on the average intensity in several areas without any detectable immunoreactivity, and intensity is then determined as the signal-to-background intensity ratio using the ImageJ software.

  3. In each group of control or treated animals, use the mean value of intensity measurements of all sections from an individual animal for statistical analysis.

  4. Express the results as the relative intensity of markers of glial or neuronal cells in the hypothalamus for each group of mice or, if two antibodies are used, the coexpression of the markers of inflammation and endoplasmic reticulum stress in the hypothalamic arcuate nucleus for each group of mice.

  5. Analyze the data using the GraphPad Prism software. Compare the two groups using the two-tailed Student’s t-test. For more than two groups, perform statistical analysis using either one-way or two-way analysis of variance (ANOVA), as appropriate, and determine statistical significance by a post-hoc analysis using the Bonferroni method, Holm-Sidak method, or Student’s t-test with P < 0.05 (Figures 1C, 2C).

Notes

  1. Alternatives

    1. If perfusion with 4% PFA is not possible, or if the perfusion was not successful, the brains should be post-fixed overnight in 4% PFA at 4°C.

    2. Brain sections can be incubated at room temperature for 2-24 h depending on the prominence of expression of the protein of interest.

    3. For double-labeled immunofluorescence, far-red fluorescent nuclear stain is recommended, but for single-labeled immunofluorescence, TOPRO should be used. Alternatively, other nuclear stains, such as DAPI or Hoechst, can be used depending on the wavelengths of the other antibodies being used.


  2. Troubleshooting

    1. If the brains are not properly cryoprotected, there is a higher chance of getting freezing artifacts.

    2. If the antibody dilution is not correct, there is a possibility that the antibody signal will be lost and there will be too much background for accurate analysis. You may need to adjust the dilutions of primary, as well as secondary antibodies, to determine the optimal concentrations required.

    3. It is recommended to be familiar with the thorough topographic anatomy of the organ under investigation, especially with the brain. There are reference books available for this task. Use the mouse brain atlas (Paxinos et al., 2001).

    4. For better resolution, do not use a spinning disk imaging microscope.

Acknowledgments

We thank the Canadian Institutes for Health Research (CIHR), Canadian Diabetes Association (CDA), and Canada Research Chairs (CRC) Program for funding this study (DDB). Scholarship support through the Banting and Best Diabetes Centre (BBDC), National Sciences and Engineering Research Council (NSERC), and the Ontario Graduate Scholarship (OGS) Program (to PSD) was much appreciated. This protocol was adapted from Dalvi et al. (2017).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed with approval from the Animal Care Committee of the University of Toronto, Canada. Standard approved housing conditions consisted of a 12 h light-dark cycle and housing with food and water ad libidum, with the recommended environmental enrichment.

References

  1. Dalvi, P. S., Chalmers, J. A., Luo, V., Han, D. Y., Wellhauser, L., Liu, Y., Tran, D. Q., Castel, J., Luquet, S., Wheeler, M. B. and Belsham, D. D. (2017). High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons. Int J Obes (Lond) 41(1): 149-158.
  2. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. J Vis Exp (65): 3564.
  3. Papouin, T. and Haydon P. G. (2018). Obtaining Acute Brain Slices. Bio-protocol 8(2): e2699.
  4. Paxinos, G. and Franklin, K. (2001). The mouse brain in stereotaxic coordinates. ISBN: 0125476361, 9780125476362. Academic Press.
  5. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.


简介

[摘要]免疫荧光是鉴定下丘脑神经元和神经胶质细胞群中特定蛋白质的可靠方法。有几种免疫荧光方案可用于检测下丘脑中的蛋白质标志物和神经肽;ħ H但是,公开的方法可以在细微的细节,可以潜在地影响该过程的最终结果而变化。在这里,我们提供了一个详细的协议,适用于薄的低温恒温器切片,该协议已成功用于针对下丘脑神经元和神经胶质细胞的关键标记的特异性抗体。我们包括有关脑t问题收集、处理、切片和标记的每一个细节,并使用最佳稀释度的抗体进行标记 瞄准的减少非特异性背景。我们的背景优化免疫染色方案已在实验室中常规使用,可以有效检测特定神经肽、神经胶质细胞以及下丘脑炎症和内质网应激的标志物。


[背景]阐明神经元和神经胶质中的蛋白质组织对于理解下丘脑功能至关重要。免疫荧光是一种强大的工具,可以研究神经元和神经胶质蛋白的表达变化,并在细胞水平上绘制激活的大脑区域。这种成像技术可以使用一抗和二抗的组合来可视化高度特异性的蛋白质(抗原)靶标。脑组织中感兴趣的抗原决定簇首先用单克隆或多克隆一抗处理,靶向宿主(小鼠、兔、鸡、大鼠、驴衍生等)中产生的感兴趣物种/蛋白质的肽。 。,是可用的),然后引导至第二抗体病房的所述的宿主初级抗体和缀合至合成或天然荧光团。Ť他抗体靶向通常标记为在脑组织切片是胶质细胞原纤维酸性蛋白(GFAP),神经元和轴突神经丝,神经肽,微管相关蛋白,血-脑屏障的蛋白,前和突触后的蛋白,髓磷脂,炎症标志物,并且与特定的疾病相关的许多抗原。其中的有用荧光标记的脑组织中抗原的可视化是罗丹明,荧光素,Alexa的氟系列,和花青染料。使用各种流行的 DNA 结合染料(如TO-PRO-3 碘化物)对细胞核进行复染,然后用二抗进行处理。

该方法已被以前使用由我们的实验室来识别神经元亚群小号中涉及能量体内平衡各种下丘脑核(Dalvi等人,2017)。特别是,使用双重免疫荧光染色,我们在小鼠急性脂质输注和慢性高脂饮食喂养后,在下丘脑和下丘脑弓状核中鉴定了 GFAP 和炎症标志物肿瘤坏死因子 (TNF) -α ,这可能促进食欲调节神经肽的含量或表达的显着改变。为了最好的我们所知,这是通过免疫荧光在暴露于高后的小鼠下丘脑弓状核证明TNF-α诱导的首次描述-脂肪饮食。该方法可以被应用到所述IDENTIF的ication中提供可靠的抗体可用于检测下丘脑区域的炎症和内质网(ER)应力的其它标志物。此协议的细节用于染色冷冻脑的冷冻切片范围从20到30μ的一般化过程米的厚度。图1和2呈现在这个协议是共焦图象的是揭示了GFAP的急性与脂质或长期用治疗的小鼠的25个μM的脑切片的表达和TNF-α在丘脑弓状核一个高-脂肪饮食(Dalvi等。, 2017)。





图1.影响急性的我在第24小时后输注C57BL / 6小鼠TNF-α,炎症标记物的丘脑弓状核(ARC)活性星形细胞增生和表达ntralipid治疗。甲- B.代表性图像示出了用于免疫的表达GFAP(绿色)和TNF-α(红色)下丘脑ARC的在24小时以下急性输注用盐水(A)或脂肪乳剂(B)C57BL6小鼠。C.量化的免疫荧光强度为GFAP和TNF-α表达的ARC的C57BL / 6小鼠在第24小时以下急性输注用盐水或脂肪乳。在柱状图数据表示为的平均值±SEM(N = 5-6只动物/组); * P < 0.05 与对照相比。A 和 B 中的图像以及 C 中的图形改编自Dalvi等人。(2017)。





图2 。的效果高-高脂肪饮食(HFD,60%千卡)8周在CD-1小鼠饲喂上下丘脑弓状核(ARC)TNF-α的活性星形细胞增生和表达。一个-乙。代表性图像示出了用于免疫的表达GFAP(绿色)和TNF-α(红色)下丘脑ARC的CD-1小鼠饲喂食物(A)或一个8周HFD(B)。C.免疫荧光的定量强度GFAP和TNF-α的表达在ARC的CD-1小鼠饲喂食物或一个HFD 8周。在柱状图数据表示为的平均值±SEM(N = 5-6只动物/组); * P < 0.05 与对照相比。A 和 B 中的图像以及 C 中的图形改编自Dalvi等人。(2017)。

关键字:下丘脑, 免疫荧光, 冰冻切片, 神经肽, 胶质纤维酸性蛋白(GFAP), TNF-α, 肿瘤坏死因子-α

材料和试剂

50 毫升猎鹰管
铝箔
精致的画笔
低温恒温器刀片(Leica Biosystems,目录号:3802106-TRI-FACET/LOPRO/CTD Blade-10PK)
用于储存冷冻切片的 12 孔板(BD Falcon,Corning,目录号:353043)
用于储存冷冻切片的 24 孔板(BD Falcon,Corning,目录号:353047)
盖玻片(25 × 50 mm)(VWR International,目录号:48393-059)
SuperFrost Plus 载玻片(带正电荷)(Thermo Fisher Scientific,目录号:12-550-15)
蜡笔(PAP Pen)(Abcam,目录号:ab2601)
PBS中的新鲜4%多聚甲醛(每只小鼠75毫升)(Millipore Sigma,目录号:158127-100G)
0.9%盐水(Millipore Sigma,目录号:S8776-100ML)
PBS pH 7.4(Thermo Fisher Scientific,Gibco,目录号:10010023) 
PBS 中的 15% 和 30% 蔗糖(1 L PBS 中的300 g蔗糖可以在冰箱中储存长达一个月)(Millipore Sigma,目录号:S7903-1KG)
冷冻保护剂溶液(Millipore Sigma,目录号:G7893-500M)
组织包埋溶液(Tissue-Tek ® OCT 化合物)(Sakura ® Finetek,VWR,目录号:25608-930)
用于制备 PBS-T 的吐温 20(PBS 中的 0.06% Tween-20)(Millipore Sigma,目录号:P1379-500ML)
Triton X-100 用于制备 PBS-Triton-X100(PBS 中的 0.4% Triton-X100)(Millipore Sigma,目录号:X100-500ML)
用于制备封闭缓冲液的正常驴血清(PBS-Triton-X100 中的 5% 血清)(Abcam,目录号:ab7475)
用于制备抗体缓冲液的正常驴血清(PBS-Triton-X100 中的 2% 血清)(Abcam,目录号:ab7475)
核染色:TOPRO(TO-PRO-3 碘化物 642/661)(Life Technologies,目录号:T3605)
Pro-Long Gold 抗褪色封固剂(封固剂)(Thermo Fisher Scientific,目录号:P10144)
透明指甲油(或用于密封盖玻片的等效物)
一抗:
抗GFAP:Ç hicken衍生多克隆小鼠抗体针对GFAP(1:500稀释)(Abcam公司,目录号:ab4674)
抗TNF-α:ģ燕麦衍生的多克隆小鼠抗体对TNF-α(1:50稀释)(Santa Cruz公司,产品目录号:SC-1350)
二抗(针对获得一抗的物种的荧光标记二抗):
驴抗鸡 IgY(H+L)二抗,FITC(将与 GFAP 鸡衍生的抗小鼠一抗相互作用)(Thermo Fisher Scientific,Invitrogen,目录号:SA1-72000)
驴抗山羊 IgG(H+L)交叉吸附二抗,Alexa Fluor 568 (将与 TNF-α 山羊衍生的抗小鼠一抗相互作用)(Thermo Fishe r Scientific,Invitrogen,目录号:A-11057 )


设备


解剖刀
镊子
细剪刀
抹刀
-20°C 冰箱
成年小鼠脑切片机矩阵(Zivic Instruments,目录号:BSMAS001-1)
小鼠灌注仪(VWR,Leica Biosystems,目录号:10030-382)
低温恒温器(徕卡,型号:CM305 0S或类似)
共聚焦激光扫描显微镜((蔡司,型号:LSM 510或类似)


软件


AxioVision 3.0 成像软件(Carl Zeiss GmbH,Jena,德国)
ImageJ 软件,美国国立卫生研究院,贝塞斯达,马里兰州,美国(Schneider等,2012)
GraphPad Prism software, Inc., La Jolla, CA, USA


程序


用于免疫荧光的脑组织的分离和制备


小鼠灌注和脑提取


在灌注的早晨(或前一天),制作新鲜的 4% 多聚甲醛 (PFA)(Gage等人,2012 年)。
注意:该溶液新鲜制备或提前不超过 72 小时制备并储存在 4°C。


使用经心脏方法,用20 毫升冷的 0.9% 盐水(或 PBS)灌注,然后是 20 毫升冷的 4% PFA(通过缓慢灌注在 5-10 分钟内最多 25 毫升)。
鼠标被灌注后,提取大脑使用手术刀,镊子,剪刀细,并且刮刀(Papouin等人,轻轻2018)放置脑入一个50 -毫升˚F爱尔康管连接用4%PFA LLED(30- 40 毫升),并在 4°C 灌注后摇动至少 6-8 小时(但不能更多)。


Cryopreserv荷兰国际集团的大脑


在 PBS 中制备 15% 和 30% 蔗糖(1 L PBS 中的 300 克蔗糖可以在冰箱中储存长达一个月)。
地方的大脑中,在50冷15%蔗糖溶液-毫升˚F在4℃爱尔康管振荡器上过夜,或直到它们下沉。
将大脑置于 50 - ml F alcon 管中的 30% 冷蔗糖溶液中,在 4°C 下在摇床上放置 2-3 天或直到它们下沉。大脑应在冷冻后一周内在低温恒温器上切割(如下所述)。


冷冻的大脑


一旦大脑已经安顿到管的底部,将它们在异戊烷浴中快速冷冻(如下所述)。
对于下丘脑的调查,冻结整个大脑之前,就把它冠前部和后部的下丘脑,S UCH该喙(前)切只是在视交叉和尾部(后)切只是在端部下丘脑(下丘脑和脑桥之间)。使用鼠标脑图谱(Paxinos等人,2001)和小鼠脑切片机矩阵到定向的大脑解剖和以准确地切割大脑。大脑的中间切割部分将包含下丘脑。
找一个小容器,用干冰装满一半。添加异戊烷以覆盖冰。将装有一些异戊烷的小烧杯放在浴缸中间。然后将每个大脑的三个切下的部分一个一个地放入烧杯中冷冻。它们应该在 10-15 秒内变白。
冷冻后,用铝箔将大脑包裹起来,贴上标签,然后在-80°C 下放置直至准备好切片。


大脑切片


Ñ OTE :脑切片可在12或24孔板或直接在载玻片上进行任一收集,取决于感兴趣的区域,时间可用性,和抗体的成本。12 孔或 24 孔板中的切片可在 -20°C 下储存直至进一步使用,但载玻片上的切片需要在收集后立即处理。如果在处理很多路段,最好使用24孔板处理(但此将需要更多的抗体)。但是,如果只处理几个部分,请使用载玻片以减少抗体用量。


提前打开低温恒温器 (Leica CM-3050-S)。
Ñ OTE :该机器需要大约两小时内冷却至-2 4 ℃。确保在继续之前达到此温度。在整个切片过程中,确保机柜温度保持在 -18°C 和 -2 4 °C 之间。的温度范围,可能需要,作为用于低温恒温器切片的最佳温度取决于组织的性质,无论是组织已经被新鲜冷冻或预先固定的,随后冷冻保护,所述的型低温恒温器,以及良好灌注是如何工作的,等。如果切割出现问题,可以升高/降低温度。特别地,如果存在与预先固定的脑切片的一个问题,尝试在切割一个较低的温度下,作为前固定大脑需要非常低的温度。


将大脑带到干冰上的低温恒温器。如果只需要下丘脑的部分,则只取出与下丘脑相对应的大脑的中间切口部分。
ñ OTE :工作快速或大脑会变形。将一对钳子放在干冰上,并带有大脑。


用乙醇清洁低温恒温器的盖子和平台。
ñ OTE :对平台的需求侧杠杆能够提升对你解开平台。


用嵌入溶液(OCT 化合物)从低温恒温器中取出组织嵌入平台 ( OCT 模具) 。用溶液覆盖平台顶部,以螺旋运动方式移动,然后将平台放在干冰上。
使用冷钳,快速将大脑转移到平台上,其尾端坐在上面,自由的喙端朝上。然后在大脑周围和顶部添加更多的嵌入解决方案以覆盖它。将它们放在干冰上,直到溶液开始冻结(会变成白色)。放置在平台上的孔的低温恒温器的内部的左侧(图3A,3B) 。
注意:另一种常用方法是在切片前将组织在嵌入平台上的嵌入溶液中预冷冻(即 OCT 模具内的 OCT 化合物)并在 -80 ° C 下储存。但是,该方法尚未在我们实验室进行测试,所以我们无法预测预嵌入和储存在-80 ° C 对蛋白质染色的影响。


一旦所有的大脑已经被移动到机器,盖上盖子,离开它了大约一个小时,因此大脑和低温切片机刀片达到相同的温度。
一旦大脑冻结,使用舞台侧面的黑色手柄放置刀片。
ñ OTE :要格外小心周围的低温恒温器叶片,因为它们是危险的锐利。


标记 24 孔板的盖子和侧面(包括样品厚度、日期等)。使用较大的巴斯德吸管将冷冻保护剂溶液至少填充一半。每个小鼠大脑使用一个 24 孔板。
将带有样品的嵌入平台锁定到前支架中并调整到位。
ñ OTE :您可以通过按下按钮在低温恒温器的前面,而不是仅仅转动拨盘移动整个设备靠近叶片。


确保使用低温恒温器上的刻度盘将机器设置为所需的厚度。
Ñ OTE :20-30μM厚度通常用于免疫荧光。


继续对大脑进行切片(图 3C)。使用细漆刷将部分转移到 24 孔板(图 3D、3E)。
ñ OTE :您可以放弃前几片,因为它们可能会由刚刚安装介质,而不是脑组织。


一旦大脑变得可见,将切片放置在含有冷冻保护剂的 24 孔板内的连续井中(图 3E、3F)。
ñ OTE小号:


使用鼠标脑图谱(Paxinos等人,定向的解剖2001年)的大脑(下丘脑)切片; 这需要实践。首先,两个侧脑室出现在切片开始处,随后出现第三个脑室,每侧都有下丘脑。
在成年小鼠,下丘脑是约5毫米长,这样将有大约250个切片用20μ米厚度和大约166个切片用30μ米的厚度。因此,如果在 24 孔板的每个孔中依次收集脑切片,则每个孔将包含来自整个下丘脑的 6-10 个切片。
或者,使用图谱,可以在 24 孔板的每个孔中依次收集一定数量的大脑切片(即大约 6-10 个切片)。必须注意如何收集切片以避免在处理免疫荧光时混淆。
尝试以从低温恒温器把它们转移时到所述板,以避免损坏最小化在电刷和部分之间的接触面积。另外,尽量以降低部分插入的舒服,所以中心,以避免出现与双方接触。
完成后,用保鲜膜盖住盘子并储存在 -20°C 的冰箱中。
ñ OTE :一定要清洗的低温恒温器,使用后嵌入平台(最重要的卸下刀片,以保持其寿命)。




图 3. 将小鼠大脑切片并将切片转移到 24 孔板中的冷冻保护剂溶液的步骤。A.组织包埋平台(OCT霉菌)使用,该平台的顶小号覆盖有溶液以螺旋运动的方式移动,并且平台被放置在干冰。使用冷镊子,大脑被迅速转移到平台上,其尾端坐在上面,嘴端自由端朝上。在大脑周围和顶部添加更多的嵌入溶液以充分覆盖它;B.上面有大脑冻结的平台留在干冰上,直到溶液开始冻结(将变成白色)。将所有大脑移入低温恒温器后,关闭低温恒温器的盖子,将大脑留在里面一个小时,使大脑和冷冻切片机刀片达到相同的温度;C.在低温恒温器上使用所需的厚度(20-30 µM 厚度通常用于免疫荧光),对脑组织进行切片;D 和 E.使用细画笔,将切下的冷冻脑切片转移到装有冷冻保护剂溶液的 24 孔板中;F.一个 24 孔板的单个孔的放大图像,里面装满了冷冻保护剂溶液和内部漂浮的大脑切片。


脑切片标记:用一抗和二抗阻断和孵育
从 -20°C 冰箱中取出含有大脑切片的24 孔板(每个 24 孔板含有冷冻保护剂中的单个小鼠的脑切片)。取一个新的 24 孔板,用 PBS-T 填充所有孔(图 4A)。将大脑切片从单个小鼠的一口或两口井转移到装有PBS-T的新 24 孔板的一个单独的井中(图 4B)。使用精细的画笔将大脑部分从冷冻保护剂转移到 PBS-T。在从防冻剂PBS-T传输会造成的扩散冷冻保护剂和包埋剂,和大脑切片将蔓延。
                            Ñ OTE :准备至少2个孔的第一抗体的阴性对照。这为成像提供了更多控制部分(这些部分在连续频繁洗涤过程中很容易损坏)(图 4B)。




图 4.将储存在 24 孔板中的冷冻保护剂溶液中的大脑切片转移到新的 24 孔板中,在 PBS 中含有 0.06% Tween-20(PBS-T)。A.准备好新的24孔板,内装PBS-T,并准备好细刷子和用清水清洗刷子的容器;B.取出所有含有浸入单个小鼠冷冻保护剂中的脑切片的24 孔板,并将来自单个小鼠的一个或两个孔的脑切片转移到新的 24 孔板的一个单独的孔中,其中填充有 PBS- T。精细画笔用于将大脑切片从冷冻保护剂转移到 PBS-T。使用 1 至 3 只小鼠的脑切片制备至少 2 个用于初级抗体阴性对照的孔。


洗涤4 ×用PBS-T,每次洗涤15分钟回转平台上。对于“洗涤”步骤,每次使用细画笔将大脑切片转移到充满 PBS-T 的新井中(参见视频 1)。对阴性对照大脑部分使用单独的刷子,并首先用这些部分开始清洗,以避免任何污染。




视频 1. 在“清洗”步骤中使用细刷将大脑切片转移到新孔中。


Ñ OTE :适当地固定在板和监测速度-如果速度迅速增大,该板可以被如果没有适当的安全移位。


在洗涤之间È S:
解冻血清等分试样。血清应与二抗的宿主物种相同。
注意:如果两种二抗的宿主物种都是驴,则使用驴血清


使用PBS-T准备封闭缓冲液。
准备额外的封闭缓冲液,因为它可以进一步稀释以制备一抗。
为了封闭组织中的潜在背景,每孔使用 1 ml 封闭溶液,剩余的可以稀释并用于制备一抗。
转移的脑切片成用于最小的封闭缓冲液孵育在室温下2小时erature或最佳过夜,在4℃。 
Ñ OTE :10%小号在erum PBS-T也可用于阻断所述组织,但最少1个小时。


阻断后,将脑切片转移到抗体缓冲液中,并适当稀释一抗。 
Ñ OTE :稀释的p rimary抗体可以是混合在一起并同时温育。在 24 孔板的每个孔中加入 0.5 至 1.0 ml 抗体缓冲液。使用较少的抗体将降低成本。


在 4°C 下孵育 12-24小时。
洗涤4 × ,用PBS-T缓冲液,每次洗涤15分钟,在室温下在回转平台上。
Ñ OTE :启动与第一抗体的阴性对照切片先与初级抗体避免污染。


用 5% 的阻塞缓冲液阻塞部分 1 小时以减少背景。
Ñ OTE :它是可以接受的,如果需要跳过这一步,作为组织应该已经被充分阻断。


转印部与抗体缓冲的二次抗体和核染色的适当稀释。
ñ OTE小号:


通常情况下,1:500稀释,用于FITC二抗,和1:1 ,000稀释用于的Alexa Fluor二抗体和核染色(TOPRO) 。
使用每组中的一些部分作为阴性二级抗体控制s 。用这些部分制作一个单独的 24 孔板,并使用单独的细画笔避免任何二次抗体的污染。
在室温下孵育 1 小时或在 4°C 下孵育过夜。
Ñ OTE :盖与铝箔避免了荧光标记的漂白。


洗涤4 × ,用PBS-T以下次级抗体一个回转平台上温育,每次洗涤15分钟。
Ñ OTE :盖与铝箔时,能够避免所述荧光团的漂白。

最后一次洗涤后,转移的部分,以PBS,慢慢地安装到切片用细刷子正电荷防滑梯。
Ñ OTE :尽可能减少切片环境照明的曝光,并且在可能时保持箔盖过的章节。将幻灯片放在带盖子的幻灯片支架盒内, 以避免将大脑部分暴露在光线下。可以在载玻片上完成一抗和二抗偶联的整个过程。将切片放在载玻片上后,用蜡笔将其圈起来,然后按照上述程序染色载玻片。这将使用更少的试剂和削减成本substan牛逼ially只有几片。


等到载玻片上的所有部分都变干。
在 4°C 的离心机中,以 1200 rpm 的速度旋转抗褪色封固剂 10 分钟,使任何固体颗粒沉淀在试管底部,不会产生任何背景信号。
免除了微量UM量的抗褪色安装媒体和安装盖玻片。
在室温或 4°C 下在黑暗中干燥 24 小时,并用透明指甲油密封边缘。
从配备彩色数码相机和 AxioVision 3.0 成像软件的蔡司 LSM 510 共焦直立显微镜上从免疫染色切片中捕获图像。使用相同的曝光时间捕获荧光图像,而不会使像素强度饱和(图 1A、1B、2A、2B)。


              数据分析


使用的ImageJ软件(施耐德等人,2012)来测量免疫荧光(光密度)的一种方式,使得样品没有识别到评估器的强度(盲法)。
为了测量荧光强度,背景是基于在几个方面的平均强度而没有任何可检测的免疫反应性,首先确定,然后强度被确定为信号-到-使用背景强度比的ImageJ软件。
在每组控制或治疗动物中,使用来自单个动物的所有部分的强度测量值的平均值进行统计分析。
表达结果作为所述神经胶质或神经元细胞的标记的下丘脑中的每组小鼠的或相对强度,如果两种抗体被使用时,炎症和内质网应激的标记物的共表达中下丘脑弓状核为每个组的老鼠。
分析了使用GraphPad Prism软件数据。使用双尾学生t检验比较两组。对于两个以上的组,即使用单向或方差分析(ANOVA)的双向分析,适当进行统计分析,并确定一统计显着性事后使用Bonferroni方法,Holm的-Sidak方法分析,或学生的吨-test与P < 0.05(图小号1C,2C)。


笔记


备择方案
如果无法使用 4% PFA 进行灌注,或者灌注不成功,则大脑应在 4% PFA 中于 4°C 下固定过夜。
脑切片可以在室温下孵育 2-24 小时,具体取决于感兴趣的蛋白质表达的突出程度。
对于双标记免疫荧光,推荐使用远红荧光核染色,但对于单标记免疫荧光,应使用TOPRO 。或者,根据所使用的其他抗体的波长,可以使用其他核染色剂,例如 DAPI 或 Hoechst。


故障排除
如果大脑不能正常冷冻保护,有一个越来越冻结人为效应的几率较高。
如果该抗体稀释是不正确的,有一种可能性,即抗体信号将会丢失,将会有准确的分析太多的背景。您可能需要调整一抗和二抗的稀释度,以确定所需的最佳浓度。
建议熟悉所研究器官的完整地形解剖学,尤其是大脑。有可用于此任务的参考书。使用小鼠脑图谱(Paxinos等,2001)。
为了获得更好的分辨率,请勿使用旋转圆盘成像显微镜。


致谢


我们感谢加拿大卫生研究院研究(CIHR),加拿大糖尿病协会(CDA) ,和加拿大研究主席(CRC)计划资助这项研究(DDB)。非常感谢班廷和最佳糖尿病中心 (BBDC)、国家科学与工程研究委员会 (NSERC) 和安大略研究生奖学金 (OGS) 计划(PSD)提供的奖学金支持。该协议改编自 Dalvi等人。(2017)。


利益争夺


该作者声明没有竞争利益。


伦理


所有的动物实验都进行批准从加拿大多伦多大学的动物护理委员会。标准核定住房条件包括的12小时光暗周期和住房食物和水的广告libidum ,与建议的环境富集。


参考


Dalvi, PS , Chalmers, JA , Luo, V. , Han, DY , Wellhauser, L. , Liu, Y. , Tran, DQ , Castel, J. , Luquet, S. , Wheeler, MB和Belsham, DD (2017) )。高脂肪诱导下丘脑的急性和慢性炎症:高脂肪饮食、棕榈酸酯和 TNF-α 对调节食欲的 NPY 神经元的影响。Int J Obes(伦敦)41(1):149-158。
Gage, GJ, Kipke, DR 和 Shain, W. (2012)。啮齿动物的全动物灌注固定。J V是E xp (65): 3564。
Papouin, T. 和 Haydon PG (2018) 。 获得急性脑切片。生物协议8(2):e2699。
Paxinos, G. 和 Franklin, K. (2001) 。立体坐标中的小鼠大脑。国际标准书号:0125476361、9780125476362 。学术出版社。
施耐德,加利福尼亚州,拉斯班德,WS和 Eliceiri,KW (2012 年)。NIH Image to ImageJ:25 年的图像分析。Nat 方法9(7): 671-675。
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引用:Dalvi, P. S. and Belsham, D. D. (2021). Immunofluorescence of GFAP and TNF-α in the Mouse Hypothalamus. Bio-protocol 11(13): e4078. DOI: 10.21769/BioProtoc.4078.
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