Diaminobenzidine dab

Unstained formalin-fixed, paraffin-embedded (FFPE) human cerebral meningeal 5 μm sections were obtained from Amsbio. For antigen retrieval, slides were incubated in pH 6.0 buffer (Reveal Decloaking reagent, Biocare Medical) at 95–98°C for 30 minutes followed by a cool down period. Subsequent steps were then performed using an immunohistochemical staining platform (Intellipath, Biocare). Slides were immersed in a 3% hydrogen peroxide solution (Peroxidazed, Biocare) for 10 minutes, rinsed with TBST and blocked using a serum-free solution (Background Sniper, Biocare Medical, Concord, CA) for 10 minutes. The blocking solution was removed and the slides were incubated with human CXCL12/SDF-1 antibody (Clone D8G6H; Cell Signaling Technology) or IgG isotype control antibody (Cell Signaling Technology, #3900) 2.7 μg/mL in 10% blocking solution/90% TBST for 60 minutes at room temperature. Slides were then washed, and detection was performed using the Novocastra Novolink Polymer Kit (Leica Microsystems Inc.) according to the manufacturer’s specifications. Detection was performed with diaminobenzidine (DAB; Biolegend). Finally, the slides were counterstained with CAT hematoxylin (Biocare), dehydrated, and coverslipped.