Biotinylated goat anti rabbit immunoglobulin

For Glial Fibrillary Acidic Protein (GFAP) and CD11b staining, slides were subjected to alkaline antigen retrieval using 10 mM Tris solution with 0.05% Tween 20 (pH 10) and microwave heating for 15 min [27 (link)]. After slides reached room temperature, peroxidase blocking using 4% H₂O₂ was performed. Sections were blocked for 60 min in 5% goat serum. The primary antibody (GFAP rabbit antibody (Millipore, Temecula, CA, USA); CD11b, rabbit antibody (Abcam, Cambridge, MA, USA)) was diluted 1:100 for both CD11b and GFAP reactions and incubated for 1 h at 37 °C. Slides were washed and incubated with biotinylated goat anti-rabbit immunoglobulin (1:500) (Dako North America Inc.) for 30 min at room temperature [30 (link)]. The biotin reaction, staining, and counterstaining were executed as described above. All serum and antibodies were diluted in phosphate-buffered saline (PBS)-bovine serum albumin (BSA)-0.1% -Triton X100, pH 7.4.

CD11b is a general marker present in all myeloid cell lineages^{40 (link)}. For CD11b staining, selected slides were subjected to alkaline antigen retrieval with 10 mM Tris solution and 0.05% Tween 20 (pH 10) for 15 min with microwave heating^{41 (link)}. Endogenous peroxidase quenching was executed as described in the previous section. Slides were blocked for 60 min in 5% goat serum followed by incubation with rabbit anti-CD11b (Abcam) diluted 1:100 and incubated for 1 h at 37 °C. The secondary antibody was a biotinylated goat anti-rabbit immunoglobulin (Dako) diluted 1:500 and incubated for 30 min at room temperature^{42 (link)}. The biotin reaction, staining, and counterstaining were executed as described in the previous section. All serum and antibodies were diluted in PBS with 1% BSA and 0.1% Triton X-100, pH 7.4.