Rhodamine red x anti mouse igg

Mice were transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were dissected, postfixed overnight at 4 °C in the same fixatives, and immersed overnight at 4 °C in PBS containing 30% sucrose. The brain specimens were then embedded in Tissue-Tek OCT compound (Sakura Finetek) and kept frozen at -80 °C until use. Transverse 20-µm sections of cervical spinal cord were prepared using a cryostat (CM1510S, Leica Biosystems). For immunohistochemical analysis, the sections were rinsed in PBS, blocked with 5% bovine serum albumin and 0.3% Triton X-100 in PBS for 1 hour at room temperature, and then incubated with a primary antibody overnight at 4 °C. The primary antibody was rabbit anti-protein kinase Cγ (PKCγ; 1:500, sc-211, RRID: AB_632234, Santa Cruz Biotechnology), mouse anti-NeuN (1:2,000, MAB377, RRID: AB_2298772, Millipore), or rabbit anti-c-Fos (1:500, 2250S, RRID: AB_2247211, Cell Signaling Technology). The sections were washed in PBS and incubated with secondary antibodies for 1 hour at room temperature. Rhodamine Red-X anti-mouse IgG (1:1,000, 715-295-151, RRID: AB_2340832, Jackson ImmunoResearch Labs) or Alexa Fluor 488 anti-rabbit IgG (1:1,000, A-21206, RRID: AB_141708, Molecular Probes) was used as a secondary antibody. All images were acquired using fluorescent microscopes (BZ-X710, Keyence; ApoTome.2, Zeiss).