Mouse smi 32 antibody for neurofilament

After transcardial perfusion with cold 4% paraformaldehyde, the retina was carefully dissected, blocked in 4% donkey serum in 0.1% triton/PBS, incubated in primary antibody at 4°C for 3-5 days, and stained with secondary antibody at 4°C. Mouse SMI-32 antibody for neurofilament (Covance) was used at a 1:2000 dilution. Multiple tiled images taken with a confocal fluorescence microscope were assembled into a mosaic image that covered the entire retina flatmount (See brain imaging section below). Counts were performed in a blinded fashion within a rectangular region that was 0.25 mm² in area (500 m × 500 m grid) at 1.5 mm from the optic nerve. Counts were made from all four quadrants of each retina flatmount in a blinded fashion. RGC densities were calculated and compared for statistical significance using the Student's t-test.

14 micron sections of retinas were stained using standard immunostaining approach. Briefly, the sections were blocked with 4% donkey serum before being incubated in primary antibody overnight at 4°C and secondary antibody the next day. The retina sections were imaged using a confocal fluorescence microscope. The following antibodies and dilutions were utilized: mouse SMI-32 antibody for neurofilament (Covance) 1:2000, rabbit anti-protein kinase C alpha (Sigma) 1:1000, mouse anti-calretinin (Millipore) 1:1000, rabbit anti-OPTN-INT (Cayman Chemical) 1:200, and rabbit anti-OPTN-C (Cayman Chemical) 1:200.