Fluoromount mounting medium fluoromount g

The retinal slices were rehydrated with phosphate-buffered saline (PBS), followed by blocking for 1 h in a solution containing 1% bovine serum albumin (BSA), 1% horse serum, and 0.3% Triton X-100 (CAS# 9002-93-1, Amresco, Solon, OH, USA) in PBS. Glial cells were labeled with rabbit anti-glial fibrillary acidic protein (GFAP) 1:500 (Cat# 13-0300, RRID: AB_2532994, Thermo Fisher, Waltham, MA, USA). Primary antibodies were diluted in blocking solution and applied overnight at 4 °C. The sections were rinsed in PBS and incubated for 1 h at RT with the secondary antibody, goat anti-rabbit Alexa Fluor 488 1:1000 (Cat# A11034, RRID: AB_2576217, Thermo Fisher Scientific, Paisley, UK). All tissue sections were counterstained with DAPI and mounted in Fluoromount mounting medium (Fluoromount-G, Cat# 0100-01, Southern Biotech, Birmingham, AL, USA). The specificity of secondary antibodies was tested by omitting the primary antibody, which did not result in any labeling. Images were obtained with a confocal microscope (Nikon C1 Plus, Nikon Instruments, NY, USA).

A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Roche Diagnostics, Mannheim, Germany) was used for the detection of apoptotic cells. Cryosections of mouse retinal explants were cultured for 7 days, rehydrated with PBS, and permeabilized for 30 min with 0.3% Triton X-100 and 0.1% sodium citrate in PBS. The TUNEL reaction was performed for 1 h at 37 °C. The sections were rinsed in PBS, counterstained with DAPI, and mounted in Fluoromount mounting medium (Fluoromount-G, Cat# 0100-01, Southern Biotech, Birmingham, AL, USA). Confocal images were analyzed by counting TUNEL-positive nuclei for each nuclear layer of the retina over a 120 μm long stretch of the retina.