Alexa fluor 546 secondary antibody solution

Brn3a is accepted as a good marker for RGC¹⁷ (link) and was used for RGC analysis in MSC-treated and untreated EAE mice and controls thirty two days after induction. Eyes of 14 mice per group were harvested, fixed in 4% paraformaldehyde for 2 hours, and prepared for wholemount staining. Retinas were permeabilized in 0.3% Triton X-100/PBS for 6 hours followed by a freeze and thaw cycle at −80°C. One percent bovine serum albumin (BSA)/0.3% Triton X-100/PBS was used for 1 hour blocking at room temperature. Retinas were incubated with goat-anti Brn3a primary antibody (Santa Cruz, TX; diluted 1:200 in 1%BSA/0.3% Triton X-100/1% DMSO/PBS) at 4°C for 48 hours on a rocker platform. After washes in PBS, a donkey anti-goat Alexa Fluor 546 secondary antibody solution (Invitrogen, Carlsbad, CA; 1:200 in PBS) was applied for 3 hours at room temperature in the dark. Following PBS washes, retinas were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Twelve images from each retina were taken at predetermined locations peripherally (5/6), mid-peripherally (3/6), and centrally (1/6) at 20x magnification using an Olympus BX41 microscope (Olympus, Center Valley, PA). Brn3a⁺ cells were counted manually by an observer masked to the protocol using the cell counter plug in ImageJ software (National Institutes of Health, Bethesda, MD). RGC numbers were normalized to density per mm².