Hamster anti cd3

Immunohistochemistry was performed on PFA-fixed free-floating tissue sections using standard protocols. Sections were washed in Tris Buffered Saline (TBS) and blocked with 5% serum for 1 hour. Primary antibodies were diluted in 0.1% Triton X-100 and 3% serum and applied overnight at room temperature. We used the following primary antibodies: rabbit anti-GFAP at 1:3,000 (Dako), rabbit anti-S100B at 1:500 (Dako), rabbit anti-TH at 1:500 (Millipore), rabbit anti-PGP 9.5 at 1:500 (Cedarlane), hamster anti-Cd3 at 1:500, rat anti-B220 at 1:500, rat antiCd31 at 1:100 (BD), biotinalayted goat anti-IgM 1:500 (ThermoFisher). The following day, the sections were rinsed extensively with TBS and incubated with a fluorescent secondary antibody for 5 hours. Secondary antibodies were all used at 1:500. To ensure that there was no non-specific binding of secondary antibodies, control sections were incubated with secondary antibody alone. With the exception of the S100B antibody, all primary antibodies yielded the expected morphology and no significant background staining. The S100B antibody exhibits known weak cross-reactivity with S100A in macrophages, which is easily distinguished in the spleen by weaker staining and a macrophage morphology. Sections were washed in TBS then wet-mounted with Vectashield hard set mounting media with DAPI (Vector Labs) and coverslipped.