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

Abstract

Source: Lani-Louzada, R., et al. Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022).

This video demonstrates the quantification of retinal ganglion cells from chemically fixed rat retinas subjected to full-circle cauterization of the limbal vascular plexus. The process includes permeabilization, antibody staining, and nuclear counterstaining, followed by visualization under an epifluorescence microscope to assess the impact of elevated intraocular pressure on retinal ganglion cell viability.

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).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DAKO	Dako North America	S3023	Antifade mounting medium
LSM 510 Meta	Carl Zeiss	-	Confocal epifluorescence microscope
Monoclonal IgG1 mouse anti-Brn3a	MilliporeSigma	MAB-1585	Brn3a primary antibody solution
TO-PRO-3	Thermo Fisher Scientific	T3605	Far red-fluorescent nuclear counterstain; emission at 661 nm
Triton X-100	Sigma-Aldrich	9036-19-5	Non-ionic surfactant
IgG donkey anti-mouse antibody + Alexa Fluor 555	Thermo Fisher Scientific	A31570	Secondary antibody solution
DAPI	Thermo Fisher Scientific	28718-90-3	diamidino-2-phenylindole; blue fluorescent nuclear counterstain; emission at 452±3 nm