A1rsi confocal microscope

Cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized and blocked in 0.1% Triton X-100 in PBS with 3% bovine serum albumin and 3% normal goat serum and for 1 hour at room temperature, and then incubated with primary antibody overnight at 4 °C. Primary antibodies used were Brn3a (Santa Cruz, TX, 1:200), βIII-tubulin (Thermo Scientific, MA 1:500), GFAP (Sigma-Aldrich, MO, 1:500), ASMase H181 (Santa Cruz, TX, 1:500). Cells were incubated with corresponding secondary antibodies for 2 hours at room temperature. DAPI (ThermoFisher Scientific, MA, 1:1000) was used to stain nuclei. Fluorescence images were acquired with a Nikon A1Rsi confocal microscope with 20x and 60x objective lenses (Fig.4) and Leica confocal microscope with 20x objective lens (Fig.3). We found two types of cells after purification with Thy 1.2 coated microbeads. The majority of cells are RGCs, on the basis of their expressing Brn3a and βIII-tubulin. Some cells also express Thy 1.2 but display distinct morphology: the size of their nuclei are 2-4 time larger than RGC nuclei, do not express Brn3a or βIII-tubulin, and do not have typical neurites. Only Brn3a and βIII-tubulin positively-stained cells were used for assessing RGC responses in this study.