Retinal Immunohistochemistry: Visualizing Cell Markers

As previously described^{38 (link),39} , eyes were harvested and fixed in 4% PFA in 1× PBS for 90 min. Retinas were dissected free from the optic cup and blocked in 10% horse serum, 0.4% Triton™ X-100 (Fisher scientific, 9002-93-1) in 1× PBS overnight at 4 °C. Retinas were then incubated at 4 °C for 3 days in primary antibodies (Table 1) diluted in 10% horse serum, 0.4% Triton™ X-100 in 1× PBS. Retinas were then washed and incubated for 24 h at 4 °C in secondary antibodies (Table 1) diluted in 1× PBS. Retinas were washed and mounted on microscope slides ganglion cell layer-up in Flourogel in TRIS buffer (Electron Microscopy Sciences, 17985-11).Table 1

Summary of antibodies

	Concentration	Company
Primary antibody (catalog #)
Rabbit anti-cCASP3 (AF835)	1:1000	R&D Systems
Rabbit anti-RBPMS (GTX118619)	1:250	GeneTex
Guinea pig anti-RBPMS (1832-RBPMS)	1:250	PhosphoSolutions
Secondary antibody (catalog #)
Donkey anti-rabbit 555 (A31572)	1:1000	ThermoFisher
Donkey anti-guinea pig 647 (706-605-148)	1:1000	Jackson ImmunoResearch

cCASP3 cleaved caspase 3, RBPMS RNA binding protein, mRNA processing factor