Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging

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.

    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.

    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.

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