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

Our research scope is to use the mouse as an animal model for studies of glaucoma to precise assess the condition of glaucoma in the mouse retina. We need to determine how many retina ganglion cells are in the retina. There are around 50, 000 retina ganglion cells in an entire mouse retina.

If we count them manually, it may take up to eight to 10 hours. Automatic counting methods can get it done in minutes and save people lots of time and effort. In the future in our lab, we are focused on glaucoma treatment study.

We are going to use different mouse models for glaucoma to test a variety of treatment methods. The method described in this protocol is particularly useful for treatment evaluation. After euthanizing the mouse using cervical dislocation for enucleation and eyeball processing, mark the edge of the cornea on the dorsal side of the eyeball using a blue marker to ensure orientation during dissection.

Using scissors, remove residual muscle tissues attached to the eyeball. Then, transfer the enucleated eyeball into a two milliliter round bottom microcentrifuge tube filled with PBS. Now, place the eyeball into another two milliliter tube containing freshly prepared 4%paraformaldehyde for fixation at four degrees Celsius for 10 minutes.

Under a dissecting microscope, use fine forceps to make a small incision on the cornea. Place the eyeball back into the fixative and incubate at four degrees Celsius for three hours to preserve tissue morphology. To remove the cornea and lens, using fine surgical scissors, make a small incision of approximately one millimeter around the corneal limbus, the border between the cornea and the sclera.

Carefully cut around the circumference of the cornea, excising it entirely to expose the underlying structures. Next, using fine forceps, gently grasp and carefully lift the lens from the surrounding tissues to avoid damaging the retina. For scleral incision, cut a precise slit in the sclera with fine ophthalmic scissors to facilitate subsequent dissection.

Next, for retinal separation, insert toothed forceps into the incision between the sclera and the retina. Gently grasp the edge of the sclera, avoiding excessive force to prevent tearing the tissue. Gradually enlarge the incision by moving the forceps along the boundary between the sclera and the retina.

Use the toothed part of the forceps to carefully separate the sclera from the retina without damaging it. Now, using sharp scissors, divide the retina into four roughly equal sized flaps. Gently unfold the dissected retina with a soft-bristled brush and meticulously remove debris from its surface.

After isolating the whole mount retina from the mouse, wash the retina in PBS with gentle agitation for five minutes. Immerse the washed retina in a blocking buffer for 30 minutes. After incubation, replace the blocking buffer with the primary antibody BRN3A.

Incubate the retina overnight at four degrees Celsius to allow specific antigen-antibody binding. The next day, wash the retina three times with PBS for five minutes each before incubating with Alexa 594 or Alexa 488 conjugated anti-rabbit secondary antibody for two hours to visualize target antigens. After washing the immunostained retina with PBS, gently transfer it onto a glass microscope slide.

Flatten the retina to minimize folding or distortion. Position a cover slip over the mounted retina, ensuring no air bubbles form. Download the code from the GitHub link, then, download the necessary files from the cloud disc and unzip them.

Locate the config. ini file and modify its content to point to the Yolo V5 CPU path. Then, click on userinterface.

exe to open the graphical interface. Check whether the pmse_plus. pt model file is in the root directory of drive C.If not, copy it from the waits folder of the Yolo V5 folder.

Open the cell counting software. Click on Open Image to import the image. Then, click on Run to allow the software to automatically count the cells.

The automated cell counting software accurately identified 15, 231 retinal ganglion cells in the N-methyl-D-aspartate-induced glaucoma mouse model, showing significant cell loss compared to approximately 45, 000 to 50, 000 RGCs in a normal retina.