Whole-mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

The procedure complied with the guidelines of the Association for Research in Vision and Ophthalmology for using animals in research and was approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Provincial People's Hospital. C57Bl/6J male mice (2 months old) were used in this study. Figure 1 illustrates the overall procedure described here. The details of the reagents and equipment used are listed in the Table of Materials.

1. Isolation of whole-mount retinas

1. Animal sacrifice
   1. Sacrifice the mice using cervical dislocation (following institutionally approved protocols) (Figure 2A). Prior to cervical dislocation,
      anesthetize the mice by inhaling isoflurane (3%-5% in oxygen) or by intraperitoneal injection of the ketamine/xylazine mixture.
2. Enucleation and eyeball processing
   1. Mark the edge of the cornea on the dorsal side of the eyeball with a blue marker to remember the orientation during subsequent dissection.
   2. Remove residual muscle tissues attached to the eyeball with scissors. Transfer the enucleated eyeball to a 2 mL round-bottom microcentrifuge tube filled with 1x PBS for a brief rinse (Figure 2C).
3. Fixation
   1. Transfer the eyeball to another 2 mL round-bottom microcentrifuge tube containing freshly made 4% paraformaldehyde (PFA) for fixation at 4 °C for 10 min (Figure 2D).
   2. Make a small incision (approximately 1-2 mm) on the cornea using fine scissors under a dissecting microscope.
   3. Place the eyeball back into the fixative for further fixation at 4 °C for 3 h to preserve tissue morphology (Figure 2D).
4. Removal of cornea and lens^1,9
   1. Make a small incision of approximately 1 mm around the corneal limbus, the border between the cornea and the sclera, using fine surgical scissors.
   2. Carefully cut around the circumference of the cornea, excising it entirely to expose the underlying structures.
   3. Use fine forceps to gently grasp and carefully lift the lens from the surrounding tissues to prevent damage to the retina. Maintain the structural integrity of the retina with delicate operation.
   4. Transfer the eyecup to a microcentrifuge tube for further processing.
      NOTE: Excise the cornea and extract the lens more efficiently using the "cross-slit" technique¹⁰. Make two perpendicular slits across the cornea and use forceps to extract the lens.
5. Scleral incision
   1. Cut a precise slit in the sclera with fine ophthalmic scissors to facilitate subsequent dissection steps (Figure 2J).
6. Retina separation
   1. Insert forceps with teeth into the incision between the sclera and the retina. Gently grasp the edge of the sclera, avoiding excessive force to prevent tearing the tissue.
   2. Gradually enlarge the incision by moving the forceps along the boundary between the sclera and the retina with slow, steady hand movements. Use the toothed part of the forceps to carefully separate the sclera from the retina, ensuring the retina is not damaged (Figure 2J).
7. Retina cutting
   1. Divide the retina into four roughly equal-sized flaps using sharp scissors. Ensure cuts are approximately halfway towards the optic nerve and oriented at 90° from each other.
8. Retina cleaning
   1. Gently unfold the dissected retina using a soft-bristled brush, and meticulously remove debris from its surface (Figure 2K).
9. Final fixation
   1. Transfer the isolated retina to a microcentrifuge tube containing fresh 4% PFA using a disposable Pasteur pipette. Allow fixation on ice for an additional 5 h to overnight for tissue preservation.

2. Immunostaining

1. Washing
   1. Wash the isolated whole-mount retina in 1x PBS with gentle agitation for 5 min. Discard the PBS solution. Repeat this process twice to remove residual fixative and debris.
2. Blocking
   1. Immerse the washed retina in a blocking buffer solution (1x PBS with 4% normal donkey serum (NDS) and 0.2% Triton X-100) for 30 min to prevent nonspecific antibody binding and enhance permeabilization (Figure 3A).
3. Primary antibody incubation
   1. Replace the blocking buffer with primary antibody BRN3A (diluted 1:200 in the blocking buffer). Incubate the retina overnight at 4 °C to allow specific antigen-antibody binding (Figure 3B).
4. Washing
   1. Wash the retina three times with 1x PBS for 5 min each to remove unbound primary antibodies and reduce background staining (Figure 3C).
5. Secondary antibody incubation
   1. Incubate the retina with Alexa594- or Alexa488-conjugated anti-rabbit secondary antibody (diluted 1:300) for 2 h at room temperature to visualize target antigens (Figure 3D).
6. Washing
   1. Perform three consecutive washes with 1x PBS for 5 min each to remove excess secondary antibodies and minimize nonspecific binding.
7. Mounting
   1. Gently transfer the immunostained retina onto a glass microscope slide, carefully flattening it to minimize folding or distortion.
   2. Apply a small drop of anti-fade mounting medium to the center of the retina, ensuring even distribution across the tissue.
   3. Carefully position a coverslip over the mounted retina, avoiding air bubbles (Figure 3E,F).

3. Image processing

NOTE: Import the image into the RGC automatic counting software and start counting. The number of BRN3A-positive cells for the entire retina can be obtained in minutes. Download the AutoCount software from GitHub (https://github.com/MOEMIL/Intelligent-quantifying-RGCs). Follow the detailed installation steps for the software below:

1. Download the code from the GitHub link. One will receive a file named "Intelligent-quantifying-RGCs.zip". Unzip this file.
2. Download the necessary files from the cloud disk (see Table of Materials) and unzip them. The downloaded file is named "yolov5_cpu". Unzip this package to the current folder to avoid unexpected issues.
3. Locate the Config file from step 1 and modify its content to point to the "yolov5_cpu" path.
   NOTE: For example, if the downloaded "yolov5_cpu" is placed in "D:1meyolov5_cpu_", change the "config.ini" file in step 1 to "D:/1me/yolov5_cpu" (use / instead of ). (Figure 4A).
4. Open the graphical interface by clicking on userinterface.exe. The initial loading may be slow (run as administrator) (Figure 4B).
5. After opening the software, verify whether the "pmse_plus.pt" model file is present in the root directory of drive C. If it is not present, copy it to this directory.
   NOTE: The "pmse_plus.pt" model file is located in the weights folder of the "yolov5" folder, found in the extracted Intelligent-quantifying RGCs folder (Figure 4C).
6. Prepare the images to be detected.
   NOTE: The software allows only one or five pictures to be read at a time. Reading more will result in an error.

4. Automated cell counting

1. Open the counting software (Figure 5A).
2. Click on OPEN IMAGE to import the image (Figure 5B).
3. Click on RUN to allow the software to automatically count the cells (Figure 5C).
   NOTE: The software will mark the counted cells with a red square box and display the cell count in the top right corner (Figure 5D).