Rat anti gm csf antibody

Thymus tissue samples were cryosectioned for immunohistochemistry using a Hyrax C60 cryostat (Zeiss). Thymus sections (10 μm) were fixed with 2% (wt/vol) paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4, and acetone, washed in PBS, and blocked with PBS supplemented with 0.1% Triton X-100 and 4% normal goat serum. Subsequently, sections were incubated with the following primary antibodies (diluted in blocking solution) overnight at 4 °C: rat anti-GM-CSF antibody (BD Pharmingen, clone BVD2- 21C11, 1:50), mouse anti-CD4 antibody (Biolegend, clone RPA-T4, 1:50) and rabbit anti-CD3 (NOVUS, clone SP7, 1:100). Sections were then washed in PBS and incubated with AF647-labeled goat anti-rat, AF488-labeled goat antimouse and AF555-labeled goat anti-rabbit secondary antibodies (Life Technologies, 1:500) overnight at 4 °C. Sections were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen). Fluorescence photomicrographs were captured with a SP5 Leica confocal laser scanning microscope (SP5; Leica) equipped with argon and helium lasers using the 40 × objective lens (oil immersion, NA1.25). Images were processed and merged by Imaris imaging software (Bitplane).