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Feb 2020
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Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging
用于图像采集的成年斑马鱼视网膜的整体免疫组化研究   

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Abstract

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Keywords: Photoreceptor (感光器), Müller glia (米勒神经胶质细胞), Visual system (视觉系统), Antibody staining (抗体显色), Microscopy (显微镜观察)

Background

To understand complex biological processes, morphological and histological analyses allow for practical qualitative and quantitative approaches. Immunohistochemistry is a robust technique used to visualize the expression and localization of intra- and extra-cellular proteins. Although conventional histological sectioning offers high resolution images of immunostained proteins, sectioning 3-dimensional tissues results in poor preservation of tissue architecture. In sections, complete cellular structures and a complete understanding of the 3-dimentional distribution of proteins are not readily appreciated. In contrast, whole-mount preparations provide large volume 3D information, including intact cellular structures and the spatial relationships of cells and molecules in a complex tissue. However, adapting immunohistochemistry for use with intact tissues often results in poor permeability of antibodies, resulting in incomplete labeling, and a high degree of non-specific background.


In the retina, the cell body of the radial Müller glia spans the entire thickness of the retina (Bringmann et al., 2006). Microglia, the innate immune cells in the central nervous system, with a small cell body and ramified processes, are distributed throughout the retinal parenchyma (Li et al., 2015; Silverman and Wong, 2018). In response to neuronal damage or death, Müller glia and microglia undergo significant structural remodelling. Characterizing the morphology of these two cell types provides a readout the local environment surrounding each cell type and the overall health of the retina (Nagashima et al., 2020; Silva et al., 2020).


Detergents and organic solvents are common reagents that efficiently break down the permeability barriers of cells and nuclei (Jamur and Oliver, 2010). Triton X-100 and Tween-20 are widely used non-ionic detergents, which perturb the phospholipid bilayer structure of biological membranes (Kalipatnapu and Chattopadhyay, 2005; Koley and Bard, 2010; Cheng et al., 2019). Although dimethyl sulfoxide (DMSO), a polar organic solvent, or acetone, a common fixative,has been shown to enhance permeability of cell membranes (de Ménorval et al., 2012), its utility in immunohistochemical procedures is less common. Most procedures select a single reagent of detergents or solvents for permeabilization, and the efficiency of combining more than two chemicals is not well documented.


Here we describe an improved method of whole-mount immunohistochemistry. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO diluted in blocking and antibody dilution buffers dramatically enhances immunolabeling in whole-mount retinas. We also report that a classic citrate buffer based epitope retrieval method (Shi et al., 1993) is effective for immunolabeling membrane associate proteins. For completeness, we also include a detailed protocol for isolating whole retinas from zebrafish.


Materials and Reagents

  1. Animals

    Adult Zebrafish (Danio Rerio, Zebrafish International Resource Center, Eugene, OR, 4 to 12 months)


  2. Materials

    1. Colorfrost Plus Microscope Slides (Fisher Scientific, catalog number: 12-550-18)

    2. Cover Glass, Rectangle No. 1½, 24 x 50 mm (Corning, catalog number: 2980-245)

    3. 1.5 ml Microcentrifuge Tubes, Free of detectable RNase, DNase, DNA & pyrogens (USA Scientific, catalog number: 1615-5500)

    4. 1 ml Syringe without needle (BD, REF 309659)

    5. ProLong Gold Antifade Mountant (hard-set; ThermoFisher Scientific, catalog number: P36930)


  3. Reagents
    Tissue preparation and fixation

    1. Tricaine-S/MS-222 (Syndel, Ferndale, WA)

    2. Sodium Bicarbonate (Sigma-Aldrich, catalog number: S5761-500G)

    3. Paraformaldehyde (SPI Supplies, catalog number: 02615-AB)

    4. Sodium Hydroxide/NaOH pellets (Sigma-Aldrich, catalog number: S-0899)

    5. Sodium Phosphate Monobasic Monohydrate/NaH2PO4·H2O (Millipore Sigma, catalog number: SX0710-3)

    6. Sodium Phosphate Dibasic/Na2HPO4 (Sigma-Aldrich, catalog number: S7907-1KG)

    7. Sucrose (Sigma-Aldrich, catalog number: S9378-1KG)

    8. 10x Anesthesia/Tricaine stock solution (see Recipes)

    9. Anesthesia/Tricaine working solution (see Recipes)

    10. 1.0% Sodium Bicarbonate solution (see Recipes)

    11. 40% paraformaldehyde stock (see Recipes)

    12. 4% paraformaldehyde in 0.1 M Phosphate buffer with 5% sucrose (see Recipes)

    13. 10x Phosphate Buffer stock (1.0 M, pH 7.4) (see Recipes)

    14. 0.1 M Phosphate buffer with 5% sucrose (see Recipes)


    Immunohistochemistry
    1. Sodium Citrate Dihydrate/C6H5Na3O7·2H2O (Fisher Scientific, catalog number: BP327-500)

    2. Tween 20 (Fisher Scientific, catalog number: BP337-500)

    3. Hydrochloric Acid/HCl (Fisher Scientific, catalog number: A144-500)

    4. Sodium Phosphate Monobasic Monohydrate/NaH2PO4·H2O (Millipore Sigma, catalog number: SX0710-3)

    5. Sodium Phosphate Dibasic/Na2HPO4 (Sigma-Aldrich, catalog number: S7907-1KG)

    6. Sodium Chloride/NaCl (Sigma-Aldrich, catalog number: S7653)

    7. Potassium Chloride/KCl (Sigma-Aldrich, catalog number: P3911-500G)

    8. Triton X-100 (Sigma-Aldrich, catalog number: T9284-500ML)

    9. Sodium Azide (Sigma-Aldrich, catalog number: S2002-100G)

    10. Dimethyl Sulfoxide (Sigma-Aldrich, catalog number: D8418-100ML)

    11. Goat Serum Donor Herd (Sigma-Aldrich, catalog number: G6767-500mL)

    12. Anti-mCherry antibody (rabbit polyclonal, Abcam, catalog number: 167453)

    13. Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, catalog number: A21245)

    14. Mouse monoclonal anti-ZO1 (ThermoFisher Scientific, ZO1-1A12, catalog number: 33-9100)

    15. Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Invitrogen, catalog number: A21422)

    16. Sodium Citrate Buffer (see Recipes)

    17. 10x Phosphate Buffered Saline (PBS, pH 7.4) stock (see Recipes)

    18. PBS (see Recipes)

    19. PBS with 0.5% Triton X-100 (see Recipes)

    20. 10x PBS with 1% sodium azide (see Recipes)

    21. Whole-mount IHC blocking buffer (see Recipes)

    22. Whole-mount IHC dilution buffer (see Recipes)

    23. Whole-mount IHC washing buffer (see Recipes)

Equipment

  1. Scissors, VANNAS, 8 cm STR (G) (World Precision Instruments, model: 14003-G)

  2. Tweezers Dumont #5 INOX (World Precision Instruments, model: 501985)

  3. Leica 205FA Stereomicroscope (Leica Microsystems)

  4. Fisher brand Isotemp Stirring Hotplate (ThermoFisher Scientific, model: SP88857200)

  5. Rocker

  6. 1,500 ml beaker (PYREX, model: CE-BEAK1L)

  7. LidLocks Microcentrifuge Tube Locks (VWR International, model: 14229-941)

  8. Round Microcentrifuge Floating Bubble Rack (VWR International, model: 60-86-342)

  9. Spoon Knife, Double Bevel, Angled 3.3 Blade (Hilcovision, model: 625-0743061-06)

  10. 15 ml Conical Tube (FALCON, model: 352097)

  11. Transfer Pipets (Fisher Scientific, model: 13-711-7M)

  12. Injection Needle with LuerLok Syringe 1 ml 30G ½ (BD, model: 305778)

  13. Zeiss AxioImage ZI Epifluorescent Microscope (Carl Zeiss MicroImaging LLC)

  14. Fisherbrand Nutating Mixers-Fixed speed (Fisher Scientific, model: 88-861-041)

Software

  1. ImageJ

  2. Adobe Photoshop CC 2019 (Adobe Systems)

Procedure

  1. Retinal dissection and fixation

    1. Dark adapt animals for at least 3 h prior to dissecting retinas.

      Note: Dark adaptation is crucial to separate neural retina from the retinal pigment epithelium. However, this step can be omitted when staining synaptic components, as dark adaptation can induce rapid changes in synaptic structure.

    2. In dark room under red illumination, sacrifice fish by immersion in ice cold water and cervical transection.

    3. Using stereomicroscope, microscissors and tweezers, carefully insert microscissors into the back of the globe to visualize optic nerve (Figure 1, arrows). Cut the optic nerve, gently remove the globe, and place it on paper towel (Figure 2).



      Figure 1. Enucleating the eye globe



      Figure 2. Front and back views of dissected globe


      Note: For better separation of neural retina and the retinal pigment epithelium, avoid bright lights. Dim illumination is acceptable.


    4. Using 30 G ½ Injection needle, make a small hole ventrally at the choroid fissure (Figure 3, red circle).



      Figure 3. Globe with ventral hole


      Note: If the choroid fissure is not visible, use other anatomical landmarks to distinguish the dorsal-ventral axis. The dorsal aspect of the eye has a greater amount of melanin/black pigments and fewer iridophores/shiny, silvery pigments, which are enriched ventrally (Figure 2).


    5. With microscissors, extend the ventral hole about 5 mm toward the optic nerve head for orientation.

    6. Cut circumferentially at the junction of the cornea and iris (Figure 4 left, red dashed line) with microscissors. Remove the entire anterior segment of the eye and lens (Figure 4).



      Figure 4. Removal of the anterior segment


    7. Place the eye cup into PBS and gently separate the neural retina from retinal pigment epithelium by inserting micro tweezers between two layers. The neural retina is faintly pink and contrasts with the underlying retinal pigmented epithelium (Figure 5).



      Figure 5. Lateral view of the eye cup in PBS


    8. Using microsurgical blade, make short relaxation cuts from the margin toward the center of the retina at the nasal, temporal and dorsal quadrants (Figure 6). These cuts help to flatten retinas during mounting on the glass slides.



      Figure 6. Dissected retina


    9. Place individual retinas in 1.5 ml microcentrifuge tube containing 1 ml of 4% paraformaldehyde in 0.1 M Phosphate buffer (pH 7.4) with 5% sucrose, and fix overnight at 4 °C on rotator (Figure 7).



      Figure 7. Fixation of retinas


    10. The next day, rinse retinas with 1 ml of 5% sucrose in 0.1 M Phosphate buffer (pH 7.4) 3 times for 20 min each on rotator at room temperature.


  2. Epitope retrieval (optional)

    Note: This procedure is necessary if your antibody recognizes membrane-associated proteins, such as Zonula Occludens (Figure 13; Nagashima et al., 2020) and N-Cadherin (Nagashima et al., 2017).

    1. Using hot plate, prepare a boiling water bath.

    2. Exchange 5% sucrose in 0.1 M Phosphate buffer (pH 7.4) with 500 μl of Sodium Citrate buffer.

    3. Immerse centrifuge tubes in boiling water bath for 5 to 10 min (Figure 8).

      Note: Install a LidLock on each microcentrifuge tube and use a floating rack.



      Figure 8. Epitope retrieval


    4. Remove beaker from hot plate and let it cool down for 5 min. Do not remove microcentrifuge tubes from the beaker.

    5. After 5 min, remove microcentrifuge tubes from the beaker and rinse retinas with 1 ml of PBS with 0.5% Triton X-100 for 10 min on rotator.


  3. Immunohistochemistry

    1. Remove buffer from the microcentrifuge tubes and add 500 μl of Whole-mount IHC blocking buffer. Incubate retinas with Whole-mount IHC blocking buffer at room temperature for 2 h on rotator.

    2. Dilute antibodies with Whole-mount IHC dilution buffer into optimized dilution (if unknown, start with 1:200). Make sure to centrifuge for 30 s before use.

      Note: If you need double or triple labeling, mix all primary antibodies together. If your sample has transgenes, such as GFP or mCherry, we recommend using anti-GFP, anti-dsRed or anti-mCherry antibodies to amplify signals. Optimal dilutions for anti-GFP, anti-dsRed or anti-mCherry vary depending on the type of transgene, cell types transgenes are expressed, and the expression level. We recommend starting with 1:200 if optimized dilution is unknown. Anti-GFP, anti-dsRed, or anti-Cherry is necessary for retinas following epitope retrieval.

    3. Incubate retinas with primary antibodies at room temperature overnight (NO agitation).

      Note: Add at least 100 μl of dilution buffer containing desired antibodies.

    4. Rinse retinas with 1 ml of Whole-mount IHC washing buffer 20 min three times at room temperature.

    5. Incubate retinas with secondary antibodies at room temperature overnight in the dark.

      Note: Add at least 100 μl of dilution buffer containing desired antibodies with optimized dilution (if unknown, start with 1:200). If you need double or triple labeling, mix all secondary antibodies together. If possible, avoid green-fluorescent conjugate, such as Alexa Fluor 488, since green channel often shows higher background, especially after antigen retrieval.

    6. Rinse retinas with 1 ml of Whole-mount IHC washing buffer 20 min three times at room temperature.


  4. Mounting on slide glass

    1. Using transfer pipette, transfer retina on a slide glass (Figure 9).



      Figure 9. Transferred retina on a slide glass


    2. Remove excess buffer with Kimwipe (DO NOT let retinas completely dry).

    3. Using tweezer, clean up and orient retinas in photoreceptor side down. A large ventral cut (see Step A8) serves as a landmark for orientating the retina. Add 1 to 2 drops of Prolong Gold onto cover glass. Gently place the cover glass on the retina for mounting (Figure 10).

      Note: Cure at least for 1 week at room temperature in the dark.



      Figure 10. Mounting retina on a slide glass



      Figure 11. Procedure of fixation and immunohistochemistry

Data analysis

Results and Discussions

Using the protocol described above (Figure 11), we successfully imaged Tg(gfap:egfp)mi2002 labeled Müller glia (Bernardos and Raymond, 2006; Nagashima et al., 2017 and 2020), Tg(mpeg1:mCherry)gl23-labeled microglia (Figure 12; Ellett et al., 2011 : Silva et al., 2020), Tg(-5.5sws1:egfp)kj9- or Tg(trb2:tdTomato)-labeled cone photoreceptors (Nagashima et al., 2017 ; Nunley et al., 2019), and Tg(-3.7rho:egfp)kj2-labeled rod photoreceptors (Raymond et al., 2014).

    Optical sectioning is essential for imaging immunofluorescence in thick specimens. Confocal microscopy eliminates out-of-focus signals and aids rapid three-dimentional reconstructions. Alternatively, we recommend epifluorescence microscopes equipped with structured illumination technology (Wu and Shroff, 2018) or computational clearing systems, which mathematically eliminate out-of-focus blur.



Figure 12. Tg(mpeg1:mCherry)gl23-labeled microglia in zebrafish retina

    The transgene, Tg(mpeg1:mCherry)gl23 (Ellett et al., 2011), was immuno-labeled with rabbit polyclonal anti-mCherry (1:200) and Alexa Fluor 647 Goat anti-Rabbit antibodies (1:200). Image was captured using a Leica TCS SP5 confocal microscope (Leica Microsystems). ImageJ software (https://imagej.nih.gov/ij/) was used for 3D rendering.



Figure 13. Comparison of zebrafish retinas immuno-labeled with ZO-1 without (left) and with (right) antigen retrieval

    An integral membrane protein, ZO1, is immuno-labeled with mouse monoclonal anti-ZO1 (1:200) and Alexa Fluor 555 Goat anti-Mouse IgG antibodies (1:200) without (left) or with (right) antigen retrieval (Figure 13). Images were captured with a Zeiss AxioImage ZI Epifluorescent Microscope. To create maximum projection of the Z-stack series, Adobe Photoshop CC 2019 (Adobe Systems) was used.

Recipes

  1. 10x Anesthesia/Tricaine stock solution (10 mg/ml Tricaine methanesulfonate)

    1. Add 1.0 g of Tricaine methanesulfonate (MS-222) to 100 ml of Mili-Q water

    2. Adjust pH to 7.4 using 1.0% Sodium Bicarbonate solution

    3. Make 2 ml aliquots and store frozen at -20 °C

  2. Anesthesia/Tricaine working solution

    Add 2 ml of Anesthesia/Tricaine stock solution to 100 ml zebrafish system water

  3. 1.0% Sodium Bicarbonate solution

    Add 1 g of Sodium Bicarbonate into 100 ml of Mili-Q water

  4. 40% paraformaldehyde stock

    1. Preheat 200 ml of Mili-Q water to 50-60 °C

    2. Add 200 g of paraformaldehyde to preheated water

    3. Stir well and add concentrated NaOH (NaOH pellets) until solution is clear

    4. Bring up volume to 500 ml (adjustment of pH is not necessary)

    5. Make 2 ml aliquots and store frozen

  5. 4% paraformaldehyde in 0.1 M Phosphate buffer with 5% sucrose

    1. Thaw 2 ml of 40% paraformaldehyde stock in/under hot water

    2. Add 1 g of sucrose and 2 ml of 10x Phosphate Buffer stock to 16 ml of Mili-Q water

    3. Add 2 ml of 40% paraformaldehyde stock

    Store at 4 °C, good for 2 weeks

  6. 10x Phosphate Buffer stock (1.0 M, pH 7.4)

    1. Dissolve 13 g of NaH2PO4·H2O and 55.75 g of Na2HPO4 in 350 ml of Mili-Q water

    2. Adjust pH to 7.4 and bring volume to 500 ml with Mili-Q water

    3. Autoclave for a long time storage

  7. 0.1 M Phosphate buffer with 5% sucrose

    1. Dissolve 25 g of sucrose in 400 ml of H2O

    2. Add 50 ml of 10x Phosphate buffer

    3. Adjust pH to 7.4 if needed

    4. Bring up volume to 500 ml with Mili-Q water

  8. Sodium Citrate Buffer (10 mM Sodium Citrate with 0.05% Tween 20, pH 6.0)

    1. Add 0.294 g of C6H5Na3O7·2H2O to 100 ml of Mili-Q water

    2. Adjust pH to 6.0 using 1 N HCl

    3. Add 0.05 ml of Tween 20 and mix well

    4. Store at room temperature for 3 months, or at 4 °C for longer storage

  9. 10x Phosphate Buffered Saline (PBS, pH 7.4) stock

    1. Add 2.76 g of NaH2PO4·H2O, 11.36 g of Na2HPO4, 87.6 g of NaCl, 1.87 g of KCl to 850 ml of Mili-Q water

    2. Adjust pH to 7.4

    3. Bring to volume to 1 L

    Each must be dissolved in solution before the next salt is added

  10. PBS

    1. Add 100 ml of 10x PBS stock to 900 ml of Mili-Q water

    2. Adjust pH to 7.4

  11. PBS with 0.5% Triton X-100

    1. Add 0.5 ml of Triton X-100 into 100 ml of PBS

    2. Mix well until Triton X-100 is completely dissolved

  12. 10x PBS with 1% sodium azide

    Add 0.5 g of sodium azide into 50 ml of 1x PBS

  13. Whole-mount IHC blocking buffer (10% Normal goat serum, 1% Tween-20, 1% Triton X-100, 1% DMSO in 0.1 M PBS with 0.1% sodium azide)

    1. Add 1.0 ml of 10x PBS with 1% sodium azide to 7.7 ml of Mili-Q water

    2. Using disposable syringe, add 0.1 ml of Tween-20 and 0.1 ml of Triton X-100 into PBS containing sodium azide (a)

    3. Add 100 μl of DMSO

    4. Mix well until Tween-20 and Triton X-100 are completely dissolved

    5. Add 1 ml of Normal goat serum

    6. Store at 4 °C, good for 1 week

  14. Whole-mount IHC dilution buffer (2% Normal goat serum, 1% Tween-20, 1% Triton X-100, 1% DMSO in 0.1M PBS with 0.1% sodium azide)

    1. Add 1.0 ml of 10x PBS with 1% sodium azide to 8.65 ml of Mili-Q water

    2. Using disposable syringe, add 0.1 ml of Tween-20 and 0.1 ml of Triton X-100 into PBS containing sodium azide (a)

    3. Add 100 μl of DMSO

    4. Mix well until Tween-20 and Triton X-100 are completely dissolved

    5. Add 50 μl of Normal goat serum

    6. Store at 4 °C, good for 1 week

  15. Whole-mount IHC washing buffer (PBS with 1% Tween-20, 1% Triton X-100, 1% DMSO)

    1. Add 10 ml of 10X PBS to 87 ml of Mili-Q water

    2. Using disposable syringe, add 1 ml of Tween-20 and 1 ml of Triton X-100 into PBS

    3. Add 1 ml of DMSO

    4. Stir well until Tween-20 and Triton X-100 are completely dissolved

    5. Store at 4 °C, good for 1 week

Acknowledgments

This work was supported by grants from the National Institutes of Health (NEI) - R01EY07060 and P30EYO7003 (PFH) and an unrestricted grant from the Research to Prevent Blindness, New York. Fish lines and reagents provided by ZIRC were supported by NIH-NCRR Grant P40 RR01. The authors thank Dilip Pawar for zebrafish maintenance and image acquisition.

Competing interests

The authors declare no competing interests.

Ethics

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan.

References

  1. Bernardos, R. L. and Raymond, P. A. (2006). GFAP transgenic zebrafish. Gene Expr Patterns 6(8): 1007-1013.
  2. Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S. N., Osborne, N. N. and Reichenbach, A. (2006). Müller cells in the healthy and diseased retina. Prog Retin Eye Res 25(4): 397-424.
  3. Cheng, R., Zhang, F., Li, M., Wo, X., Su, Y. W. and Wang, W. (2019). Influence of fixation and permeabilization on the mass density of single cells: A surface plasmon resonance imaging study. Front Chem 7: 588.
  4. de Ménorval, M. A., Mir, L. M., Fernandez, M. L. and Reigada, R. (2012). Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: a comparative study of experiments in silico and with cells. PLoS One 7(7): e41733.
  5. Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. and Lieschke, G. J. (2011). mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117(4): e49-e56.
  6. Jamur, M. C. and Oliver, C. (2010). Permeabilization of cell membranes. Methods Mol Biol 588: 63-66.
  7. Kalipatnapu, S. and Chattopadhyay, A. (2005). Membrane protein solubilization: recent advances and challenges in solubilization of serotonin1A receptors. IUBMB Life 57(7): 505-512.
  8. Koley, D. and Bard, A. J. (2010). Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy(SECM). Proc Natl Acad Sci U S A 107(39): 16783-16787.
  9. Li, L., Eter, N. and Heiduschka, P. (2015). The microglia in healthy and diseased retina. Exp Eye Res 136: 116-130.
  10. Nagashima, M., Hadidjojo, J., Barthel, L. K., Lubensky, D. K. and Raymond, P. A. (2017). Anisotropic Müller glial scaffolding supports a multiplex lettice mosaic of photoreceptors in zebrafish retina. Neural Dev 12(1): 20.
  11. Nagashima, M., D’Cruz, T. S., Danku, A. E., Hesse, D., Sifuentes, C., Raymond, P. A. and Hitchcock, P. F. (2020). Midkine-a is required for cell cycle progression of müller glia during neuronal regeneration. in the vertebrate retina. J Neurosci 40(6): 1232-1247.
  12. Nunley, H., Nagashima, M., Martin, K., Gonzalez, A. L., Suzuki, S. C., Norton, D., Wong, R. O. L., Raymond, P. A. and Lubensky, D. K. (2019). Defect patterns on the curved surface of fish retinae suggest mechanism of cone mosaic formation. https://doi.org/10.1101/806679
  13. Raymond, P. A., Colvin, S. M., Jabeen, Z., Nagashima, M., Barthel, L. K., Hadidjojo, J., Popova, L., Pejaver, V. R. and Lubensky, D. K. (2014). Patterning the cone mosaic array in zebrafish retina requires specification of ultraviolet-sensitive cones. PloS One 9(1): e85325.
  14. Shi, S. R., Chaiwun, B., Young, L., Cote, R. J. and Taylor, C. R. (1993). Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 41(11): 1599-1604.
  15. Silva, N. J., Nagashima, M., Li, J., Kakuk-Atkins, L., Ashrafzadeh, M., Hyde, D. R. and Hitchcock, P. F. (2020). Inflammation and matrix metalloproteinase 9(Mmp-9) regulate photoreceptor regeneration in adult zebrafish. Glia 68(7): 1445-1465.
  16. Silverman, S. M. and Wong, W. T. (2018). Microglia in the Retina: Roles in Development, Maturity, and Disease. Annu Rev Vis Sci 4: 45-77.
  17. Wu, Y. and Shroff, H. (2018). Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat Methods 15(12): 1011-1019.

简介

Abstra CT ]免疫组织化学是一种广泛使用的技术来检验表达和蛋白质的亚细胞定位。该技术依赖于抗体的特异性,并且需要抗体充分渗透到组织中。对于厚的标本,例如胚胎和其他整装样品,后者尤其具有挑战性。在这里,我们描述了一种用于视网膜整装制剂的免疫组织化学的改进方法。我们报告说,三种试剂鸡尾酒,曲通X - 封闭和抗体稀释缓冲液中的100,Tween-20和DMSO强烈增强了成年斑马鱼整个视网膜中的免疫标记。此外,我们建立了在整个视网膜组织中,基于柠檬酸盐缓冲液的经典表位检索方法,可以有效地免疫标记膜相关蛋白。总的来说,这种简单的修饰可以对视网膜整装中的蛋白质进行精确且可重复的免疫标记。


[背景]为了理解复杂的生物过程,形态学和组织学分析能够进行切合实际的定性和quantitativ ë接近。免疫组织化学是一种用于可视化细胞内和细胞外蛋白表达和定位的强大技术。尽管常规组织学切片可提供高分辨率的免疫染色蛋白图像,但对3维组织进行切片会导致组织结构保存不佳。在各节中,不容易理解完整的细胞结构和对蛋白质3维分布的完整理解。相反,整装制剂可提供大量3D信息,包括完整的细胞结构以及复杂组织中细胞和分子的空间关系。然而,使免疫组织化学适合与完整组织一起使用通常会导致抗体的渗透性差,导致标记不完全和高度的非特异性背景。

在视网膜,径向M的细胞体ü米勒胶质细胞跨度视网膜的整个厚度(Bringmann等人,2006)。小胶质细胞是中枢神经系统的先天免疫细胞,具有小的细胞体和分叉的过程,分布在整个视网膜实质中(Li等人,2015; Silverman和Wong,2018)。为了响应神经元损伤或死亡,男ü米勒胶质细胞和小胶质细胞进行显著结构重构。表征这两种细胞类型的形态可提供每种细胞类型周围的局部环境以及视网膜的整体健康状况的读数(Nagashima等,2020; Silva等,2020)。

洗涤剂和有机溶剂是有效打破细胞和细胞核渗透屏障的常用试剂(Jamur和Oliver ,2010)。Triton X-100和Tween-20是广泛使用的非离子型去污剂,它们会扰动生物膜的磷脂双层结构(Kalipatnapu和Chattopadhyay,2005 ; Koley和Bard,2010; Cheng等人2019) 。尽管二甲亚砜(DMSO),极性有机溶剂,或丙酮,一个共同的固定剂,已被证明能提高细胞膜的通透性(DEMénorval等人,2012) ,其在免疫组织化学程序实用程序是不常见的。大多数程序只选择一种去污剂或溶剂的试剂进行通透性,而且两种以上化学品混合的效率还没有很好的证明。

在这里,我们描述了一种完整的免疫组织化学的改进方法。我们报告说,在封闭液和抗体稀释液中稀释的三种试剂(Triton X - 100,Tween-20和DMSO)的混合物极大地增强了整个视网膜的免疫标记。我们还报道了基于经典柠檬酸盐缓冲液的表位检索方法(Shi等,1993)对于免疫标记膜缔合蛋白是有效的。为了完整起见,我们还包括从斑马鱼中分离整个视网膜的详细协议。

关键字:感光器, 米勒神经胶质细胞, 视觉系统, 抗体显色, 显微镜观察


材料和试剂
 
动物
成年斑马鱼(Danio Rerio,斑马鱼国际资源中心,俄勒冈州尤金)4至12个月
 
用料
1. Colorfrost Plus显微镜载玻片(Fisher Scientific,目录号:12-550-18)      
2.矩形玻璃盖1       ½,24 x 50毫米(Corning,目录号:2980-245)
3. 1.5 ml微量离心管,不含可检测的RNase,DNase,DNA和热原(美国科学公司,目录号:1615-5500)      
4. 1毫升无针注射器(BD,REF 309659)      
5. ProLong黄金防褪色固定剂(固化; ThermoFisher Scientific,目录号:P36930)      
 
试剂种类
组织准备和固定
Tricaine-S / MS-222(华盛顿州芬代尔Syndel)
碳酸氢钠(Sigma - Aldrich,目录号:S5761-500G)
多聚甲醛(SPI Supplies,目录号:02615-AB) 
氢氧化钠/ NaOH粒料(Sigma - Aldrich,目录号:S-0899)
一水磷酸钠/ NaH 2 PO 4· H 2 O(Millipore Sigma,目录号:SX0710-3)
磷酸氢二钠/ Na 2 HPO 4 (Sigma - Aldrich,目录号:S7907-1KG)
蔗糖(Sigma - Aldrich,目录号:S9378-1KG)
10倍麻醉/三卡因储备溶液(请参阅食谱)
麻醉/三卡因工作溶液(请参阅食谱)
1.0%碳酸氢钠溶液(请参阅食谱)
40%多聚甲醛库存(请参阅食谱)
在0.1 M磷酸盐缓冲液和5%蔗糖中的4%低聚甲醛(请参阅食谱)
10x磷酸盐缓冲液储备液(1.0 M,pH 7.4)(请参见配方)
具有5%蔗糖的0.1 M磷酸盐缓冲液(请参见食谱)
 
免疫组织化学
柠檬酸钠二水合物/ C 6 H 5 Na 3 O 7 · 2H 2 O(Fisher Scientific,目录号:BP327-500)
吐温20(Fisher Scientific,目录号:BP337-500)
盐酸/ HCl(Fisher Scientific,目录号:A144-500)
一水磷酸二氢钠/ NaH 2 PO 4 · H 2 O(Millipore Sigma,目录号:SX0710-3)
磷酸氢二钠/ Na 2 HPO 4 (Sigma - Aldrich,目录号:S7907-1KG)
氯化钠/ NaCl(Sigma - Aldrich,目录号:S7653)
氯化钾/氯化钾(Sigma - Aldrich,目录号:P3911-500G)
海卫一X-100(Sigma - Aldrich,目录号:T9284-500ML)
叠氮化钠(Sigma - Aldrich,目录号:S2002-100G)
二甲基亚砜(Sigma - Aldrich,目录号:D8418-100ML)
山羊血清供体群(Sigma - Aldrich,目录号:G6767-500mL)
抗mCherry抗体(兔多克隆,Abcam,目录号:167453)
山羊抗兔IgG(H + L)高度交叉吸附二级抗体,Alexa Fluor 647(Invitrogen,目录号:A21245)
小鼠单克隆抗ZO1(ThermoFisher Scientific,ZO1-1A12,目录号:33-9100)
山羊抗小鼠IgG(H + L)交叉吸附二级抗体,Alexa Fluor 555(Invitrogen,目录号:A21422)
柠檬酸钠缓冲液(见配方)
10倍磷酸盐缓冲盐水(PBS,pH 7.4)储备液(请参阅食谱)
PBS(请参阅食谱)
含0.5%Triton X-100的PBS(请参阅食谱)
10x PBS和1%叠氮化钠(请参阅食谱)
整个安装的IHC阻断缓冲液(请参见配方)
完整的IHC稀释缓冲液(请参见食谱)
整个安装的IHC清洗缓冲液(请参阅配方)
 
设备
 
VANNAS剪刀,STR(G)8厘米,(World Precision Instruments,型号:14003-G)
杜蒙特镊子#5 INOX(世界精密仪器公司,型号:501985)
徕卡205FA立体显微镜(Leica Microsystems)
Fisher品牌等温搅拌炉(ThermoFisher Scientific,型号:SP88857200)
摇杆
1,500 ml烧杯(PYREX,型号:CE-BEAK1L)
LidLocks的离心管锁(VWR我国际电信联盟,型号:14229-941)
圆形微型离心机浮动泡沫架(VWR我国际电信联盟,型号:60-86-342)
汤匙刀,双斜角,弯角3.3刀片(Hilcovision,型号:625-0743061-06)
15 ml锥形管(FALCON,型号:352097)
移液器(Fisher Scientific,型号:13-711-7M)
带有LuerLok注射器的注射针1 ml 30G½ (BD,型号:305778)
蔡司AxioImage ZI荧光显微镜(Carl Zeiss MicroImaging LLC)
Fisherbrand Nutating搅拌机-固定速度(Fisher Scientific,型号:88-861-041)
 
小号oftware
 
图像
Adobe Photoshop CC 2019(Adobe系统)
 
程序
 
视网膜解剖和固定
1.在解剖视网膜之前,黑暗适应动物至少3小时。      
注意:黑暗适应对于将神经视网膜与视网膜色素上皮分开至关重要。但是,在对突触成分进行染色时可以省略此步骤,因为黑暗适应会导致突触结构快速变化。
2.在红色照明的暗室中,将鱼浸入冰冷的水中并颈椎横断处死。      
3.使用立体显微镜,微剪刀和镊子,小心地将微剪刀插入地球仪的背面,以可视化视神经(图1,箭头)。切开视神经,轻轻取下球体,然后将其放在纸巾上(图2)。      
 


图1.摘除眼球
 


图2.解剖后的球体的前视图和后视图
 
注意:为了更好地分离神经视网膜和视网膜色素上皮,避免强光照射。昏暗的照明是可以接受的。
 
4.使用30 ģ ½注射针,在脉络膜裂使一个小孔腹侧(图3,红色圆圈)。           
 


图3.带有腹孔的地球仪
 
注意:如果脉络膜裂隙不可见,请使用其他解剖标记来区分背腹轴。眼睛的背面有大量的黑色素/黑色色素和较少的虹膜/闪亮的银色色素,这些色素在腹侧富集(图2)。
 
5.使用微剪刀,将腹侧孔向视神经头延伸约5 mm,以进行定向。      
6.用小剪刀在角膜和虹膜的交界处沿周向切口(图4左,红色虚线)。去除眼睛和晶状体的整个前段(图4)。      
 


图4.去除前段
 
7.将眼罩放入PBS中,并通过在两层之间插入微型镊子,将神经视网膜与视网膜色素上皮轻轻分开。神经视网膜微弱的粉红色,与下面的视网膜色素上皮形成对比(图5)。      
 


图5.晚于PBS眼罩的RAL视图
 
8.使用显微外科手术刀片,在鼻,颞和背象限处,从边缘向视网膜中心进行短暂的松弛切割(图6)。这些切口有助于在将玻璃片安装到玻璃片上时使视网膜变平。      
 


图6。解剖的视网膜
 
9.将单个视网膜放入1.5 ml微量离心管中,该离心管中含有1 ml 4%多聚甲醛的0.1 M磷酸盐缓冲液(pH 7.4)和5%蔗糖,并在4 °C的转子上固定过夜(图7 )。      
 


图7.视网膜固定
 
10.第二天,在室温下用1 ml的5%蔗糖在0.1 M磷酸盐缓冲液(pH 7.4)中冲洗视网膜3次,每次20分钟。   
 
表位检索(可选)
注意:如果您的抗体识别膜相关蛋白,例如Zonula Occludens(图13; Nagashima等人,2020)和N-钙黏着蛋白(Nagashima等人,2017),则此步骤是必需的。 
用热板准备沸水浴。
交换5%蔗糖的0.1 L磷酸盐缓冲液(pH为7.4)与500 μ钠升柠檬酸盐缓冲液。
将离心管浸入沸水浴中5至10分钟(图8 )。
注意:在每个微量离心管上安装一个LidLock,并使用浮动架子。
 


图8.表位检索
 
从热板上移出烧杯,使其冷却5分钟。不要从烧杯中取出微量离心管。
5分钟后,从烧杯中取出微量离心管,并在转子上用1 ml PBS和0.5%Triton X -100冲洗视网膜10分钟。
 
免疫组织化学
取下离心管缓冲液和加入500微升全安装IHC封闭缓冲液。在室温下,在转子上用全载IHC封闭缓冲液孵育视网膜2小时。
用整装IHC稀释缓冲液将抗体稀释至最佳稀释度(如果未知,则从1:200开始)。使用前,请确保离心30 s。
注意:如果您需要双重或三次标记,请将所有一抗混合在一起。如果您的样品具有转基因,例如GFP或mCherry,我们建议使用抗GFP,抗dsRed或抗mCherry抗体来放大信号。抗GFP,抗dsRed或抗mCherry的最佳稀释度取决于转基因的类型,表达转基因的细胞类型和表达水平。如果未知最佳稀释度,建议从1:200开始。表位修复后的视网膜需要抗GFP,抗dsRed或抗樱桃。
在室温下将视网膜与一抗孵育过夜(不搅动)。
注意:添加至少100μl含有所需抗体的稀释缓冲液。
在室温下,用1 ml完整安装的IHC洗涤缓冲液冲洗视网膜20分钟,每次3次。
在黑暗中于室温下将视网膜与第二抗体孵育过夜。
注意:添加至少100μl稀释缓冲液,其中含有经过优化稀释的所需抗体(如果未知,请从1:200开始)。如果需要双重或三次标记,请将所有第二抗体混合在一起。如果可能,请避免使用绿色荧光偶联物,例如Alexa Fluor 488,因为绿色通道通常显示出较高的背景,尤其是在抗原回收后。
在室温下,用1 ml的全挂载IHC洗涤缓冲液冲洗视网膜20分钟,每次3次。
 
安装在载玻片上
使用移液器将视网膜移至载玻片上(图9 )。
 


图9.载玻片上的Tran刺激视网膜
 
用K抹去多余的缓冲液(不要让视网膜完全干燥)。
使用镊子清理并对准光感受器的视网膜。较大的腹侧切口(请参阅步骤A8 )用作定向视网膜的界标。在盖玻片上加入1至2滴Prolong Gold。将盖玻片轻轻放在视网膜上以进行固定(图10 )。
注意:在黑暗中于室温下至少固化1周。
 


图10.中号在载玻片上ounting视网膜
 


图11.固定和免疫组织化学程序
 
d ATA分析
 
结果与讨论
使用上述协议(图11),我们成功地成像了标记为Müller胶质细胞(Tern (gfap :egfp))的mi2002 (Bernardos和Raymond ,2006; Nagashima等人,2017和2020),Tg(mpeg1:mCherry)gl23标记的小胶质细胞(图12; Ellett等,2011:Silva等,2020),Tg(-5.5sws1:egfp)kj9-或Tg(trb2:tdTomato)标记的锥感光细胞(Nagashima等,2017; Nunley等等人,2019)和Tg(-3.7rho:egfp)kj2标记的棒状感光器(Raymond等人,2014)。
 
  光学切片对于在厚样本中成像免疫荧光至关重要。共聚焦显微镜可以消除散焦信号,并有助于快速的三维重建。可替换地,我们建议落射荧光显微镜小号配备有结构化照明技术(Wu和缴费,2018)或计算结算系统小号,这在数学上消除了-的-焦模糊。
 


图12. TG(MPEG1:mCherry的)gl23 -labele d的小胶质细胞在斑马鱼视网膜
 
用兔多克隆抗mCherry(1:200)和Alexa Fluor 647山羊抗兔抗体(1:200)对转基因Tg(mpe g1:mCherry)gl23 (Ellett et al。,2011)进行免疫标记。 使用Leica TCS SP5共焦显微镜(Leica Microsystems)捕获图像。使用ImageJ软件(https://imagej.nih.gov/ij/)进行3D渲染。
 


图13.用(未)(左)和((右))抗原回收的ZO-1免疫标记的斑马鱼视网膜的比较
 
完整的膜蛋白ZO1用小鼠单克隆抗ZO1(1:200)和Alexa Fluor 555山羊抗小鼠IgG抗体(1:200)进行了免疫标记,而没有(左)或(右)抗原回收(图13)。用Zeiss AxioImage ZI Epif荧光显微镜捕获图像。为了创建Z-stack系列的最大投影,使用了Adobe Photoshop CC 2019(Adobe Systems)。
 
菜谱
 
10 x麻醉/大肠储备液(10 mg / ml曲卡因甲磺酸盐)
将1.0克的Tricaine甲磺酸盐(MS-222)添加到100毫升的Mili-Q水中
使用1.0%碳酸氢钠溶液将pH调节至7.4
制作2毫升等分试样,并冷冻保存在-20 °C
麻醉/三卡因工作溶液
向100 ml斑马鱼系统水中添加2 ml麻醉/三氮烷储备溶液
1.0%碳酸氢钠溶液
将1克碳酸氢钠加到100毫升Mili-Q水中
40%低聚甲醛库存
将200 ml的Mili-Q水预热至50-60 °C
在预热的水中添加200克变形甲醛
充分搅拌并加入浓NaOH(NaOH颗粒)直至溶液澄清
体积带来高达500毫升(一pH值为djustment是不必要的)
制作2毫升等分试样并冷冻保存
含5%蔗糖的0.1 M磷酸盐缓冲液中的4%多聚甲醛
在热水中/下融化2 ml 40%多聚甲醛
将1克蔗糖和2毫升10 x磷酸盐缓冲液加入16毫升Mili-Q水中
加入2 ml 40%多聚甲醛
储存在4 °C下,可保存2周
10x磷酸盐缓冲液库存(1.0 M,pH 7.4)
将13 g NaH 2 PO 4 · H 2 O和55.75 g Na 2 HPO 4溶解在350 ml Mili-Q水中
将pH调节至7.4,并用Mili-Q水调节至500 ml
高压灭菌器可长期保存
0.1 M磷酸盐缓冲液和5%蔗糖
将25克蔗糖溶于400毫升H 2 O
加入50毫升的10倍磷酸盐缓冲液
如果需要,将pH值调节至7.4
用Mili-Q水将容量调至500 ml
柠檬酸钠缓冲液(10 mM柠檬酸钠,0.05%T介于20之间,pH 6.0)
向100 ml的Mili-Q水中添加0.294 g的C 6 H 5 Na 3 O 7 · 2H 2 O
使用1 N HCl将pH调整至6.0
加入0.05 ml的Tween 20并充分混合
在室温下存放3个月,或在4 °C下存放更长的时间
10倍磷酸盐缓冲盐水(PBS,pH 7.4)
加入的NaH2.76克2 PO 4 · H ^ 2 O,11.36克的Na 2 HPO 4 87.6克NaCl,1.87克的KCl至850毫升米利-Q水
调节pH至7.4
调至1 L
每一种都必须溶解在溶液中,然后再添加下一种盐
PBS
将100毫升的10倍PBS储备液添加到900毫升的Mili-Q水中
调节pH至7.4
含0.5%Triton X-100的PBS
将0.5 ml的Triton X-100加入100 ml的PBS中
充分搅拌直到Triton X-100完全溶解
10x PBS和1%叠氮化钠
将0.5克叠氮化钠加入50毫升1x PBS中
全血IHC封闭缓冲液(10%正常山羊血清,1%Tween-20、1%Triton X-100、1%DMSO的0.1M PBS和0.1%叠氮化钠溶液)
向7.7 ml Mili-Q水中添加1.0 ml含1%叠氮化钠的10x PBS
使用一次性注射器,将0.1 ml的Tween-20和0.1 ml的Triton X-100加入含叠氮化钠(a)的PBS中
加100 μ升DMSO的
充分混合直至Tween-20和Triton X-100完全溶解
加入1毫升普通山羊血清
储存在4 °C下,可保存1周
完整的IHC稀释缓冲液(2 %正常山羊血清,1%Tween-20、1%Triton X-100、1%DMSO的0.1M PBS和0.1%叠氮化钠溶液)
将1.0 ml含1%叠氮化钠的10x PBS加入8.65 ml Mili-Q水中
使用一次性注射器,将0.1 ml的Tween-20和0.1 ml的Triton X-100加入含叠氮化钠(a)的PBS中
加入100微升的DMSO中
充分混合直至Tween-20和Triton X-100完全溶解
加入50μl正常山羊血清
储存在4 °C下,可保存1周
完整的IHC洗涤缓冲液(含1%Tween-20、1%Triton X-100、1%DMSO的PBS)
将10毫升的10X PBS加到87毫升的Mili-Q水中
使用一次性注射器,将1 ml Tween-20和1 ml Triton X-100加入PBS
加入1毫升DMSO
充分搅拌直至Tween-20和Triton X-100完全溶解
储存在4 °C下,可保存1周
 
致谢
 
这项工作得到了美国国立卫生研究院(NEI)-R01EY07060和P30EYO7003(PFH)的资助,以及纽约预防失明研究的无限制资助。ZIRC提供的鱼线和试剂由NIH-NCRR Grant P40 RR01支持。作者感谢Dilip Pawar维护斑马鱼和获取图像。
 
利益争夺
 
作者宣称没有利益冲突。
 
伦理
 
所有动物程序均由密歇根大学的机构动物护理和使用委员会(IACUC)批准。
 
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nagashima, M. and Hitchcock, P. F. (2020). Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging. Bio-protocol 10(24): e3848. DOI: 10.21769/BioProtoc.3848.
  2. Nagashima, M., D’Cruz, T. S., Danku, A. E., Hesse, D., Sifuentes, C., Raymond, P. A. and Hitchcock, P. F. (2020). Midkine-a is required for cell cycle progression of müller glia during neuronal regeneration. in the vertebrate retina. J Neurosci 40(6): 1232-1247.
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