Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.    

1. Quantification of retinal ganglion cells somas

NOTE: The following procedure is for quantification of RGC somas, based on immunohistochemical staining of retinal flat-mounts with an antibody against the brain-specific homeobox/POU domain protein 3A (Brn3a).

1. Transfer isolated retinas into a 24-well culture plate (one retina per well) containing 1 mL of 1x PBS and keep the inner retina facing up.
2. Permeabilize tissue by washing 3x for 10 min with a non-ionic surfactant 0.5% diluted in 1x PBS (0.3 mL). Then keep the tissue gently shaking in 5% bovine serum albumin (BSA) in non-ionic surfactant 2% and 1x PBS (blocking solution; 0.25 mL) for 60 min at room temperature.
3. During step 1.2, prepare the Brn3a primary antibody solution by diluting 1:200 in non-ionic surfactant 0.5% and 1x PBS plus 5% BSA, and store it at 4 °C.
4. After 60 min of tissue blocking, incubate retinas in 0.2 mL of primary antibody solution at 4 °C for 72 h with gentle shaking.
5. Wash the tissue 3x for 10 min with 1x PBS, then incubate the tissue for 2 h at room temperature in 0.2 mL of the secondary antibody solution diluted 1:750 in 1x PBS plus 5% BSA.
6. Further, incubate the tissue for 10 min in nuclear counterstain solution for fluorescent nuclei staining. Conclude immunohistochemistry with a final washing step with 1x PBS (0.3 mL) repeated 3x.
7. Transfer retinas with the aid of two small brushes onto glass microscope slides, maintaining the vitreous side up. Position the dorsal retinal quadrant up on the microscope slides. Make two more radial cuts towards the optic nerve head to delimit the other 3 quadrants (nasal, ventral, and temporal).
   NOTE: The cut dimensions are not fixed. They should not be too short that impairs an efficient flattening of the retina on the slide and should not be too long so that it reaches the optic nerve foramen and completely separates the delimited retinal quadrant from the rest of the tissue.
8. Finally, apply 0.2 mL of antifade mounting medium on a glass coverslip and place this onto the flat-mounted retina for tissue microscopic analysis. To estimate RGCs density, examine the flat mounts under a confocal epifluorescence microscope using a 40x/1.3 objective.
9. For each quadrant of the retina, take eight photos: two from the central retina (~0.9 mm from optic disc), three from mid-retina (~2.0 mm from optic disc), and three from the peripheral retina (~3.7 mm from optic disc), totaling to 32 photos per retina. Use the FIJI software to count Brn3a-positive cells and estimate the mean cell density.

Materials