Fast red nuclear stain

A cre-mediated alkaline phosphatase (AP) reporter, provided by Tudor Badea in Jeremy Nathan’s lab, was expressed in conjunction with Opn4^Cre (Ecker et al., 2010 (link)). Mice were deeply anesthetized with 30 ml/kg Avertin and then intracardially perfused with phosphate-buffered saline for 3 min followed by 40 ml of 4% paraformaldehyde. Brains and retinas were isolated and post-fixed for 40 min in 4% paraformaldehyde. Brains were mounted in 3% agarose and then cut into 200 µm sections on a vibrating microtome (Vibra-tome 1000 Plus). Tissue was heat-inactivated for overnight at 65°C. Alkaline phosphatase histochemistry was performed using NBT/BCIP tablets (Roche) for 2–4 hr in the dark with constant shaking. Tissue was washed three times with phosphate-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich). Retinas were mounted immediately and imaged. Brains were fixed 3 hr in 4% paraformaldehyde at 4°C, then counterstained with 1:5 Fast Red nuclear stain (Vector Laboratories) in water for 7 min. The sections were then dehydrated in an ethanol series, and after at least in hour in 100% ethanol, the sections were cleared in a 2:1 mixture of benzyl benzoate:benzyl alcohol (Sigma-Aldrich), mounted in glycerol, and imaged immediately. To measure cell density, we counted the number of ipRGCs, in four representative areas of each retina, and calculated the density of ipRGCs per mm².