Bz x700

The optic nerves were isolated for immunohistochemistry on day 7 after the ONC as described. The isolated optic nerves were postfixed in 4% PFA in PBS overnight. After washing with PBS, the tissues were immersed in 30% sucrose for 2 days at 48C and then embedded in OCT compound (Sakura Finetek, Torrance, CA, USA). Then, 5-lm frozen sections were cut with a cryostat. After blocking the sections in 1.0% normal goat or donkey serum plus 1.0% BSA and 0.1% Triton-X 100 in PBS, the sections of the optic nerves were incubated with the primary antibodies of mouse anti-CD68 antibody (1:500; Serotec, Oxford, UK), goat polyclonal anti-MCP-1 antibody (1:200; Santa Cruz, Dallas, TX, USA), mouse monoclonal or rabbit polyclonal anti-GFAP antibody (1:500; Merck Millipore, Billerica, MA, USA), or rabbit polyclonal anti-iNOS antibody overnight at 48C. The sections were then incubated overnight at 48C in Alexa 594 or Alexa 488 conjugated to the appropriate secondary antibodies (Invitrogen, Carlsbad, CA, USA) diluted by 1:500.
The sections were photographed with a fluorescence microscope (BZ-X700) or a confocal laser microscope (TCS SP8; Leica, Wetzlar, Germany).