Mca1957

Fixed tissues were sectioned (coronal for cerebrum and sagittal for cerebellum) at 35 μm thickness using a Leica V100S vibratome (McGlynn et al., 2004 (link)). For immunofluorescent staining, tissues were first blocked for 90 minutes at 4°C with shaking (10% goat serum, 0.02% saponin, 1× PBS). Sections were then incubated with primary antibody in diluent solution (5% normal goat serum, 0.02% saponin, 1× PBS) overnight at 4° C. Primary antibody(ies) included anti-GM2 (1:50, mouse IgM, cell culture supernatant produced in-house by Dr. K. Dobrenis using the 10–11 hybridoma line from Progenics Pharmaceuticals, Tarrytown, NY USA), anti-CD68 (1:500, rat monoclonal antibody, AbD Serotec MCA1957), anti-GFAP (1:1000, rabbit polyclonal antibody, Sigma G9269), and anti-Calbindin-D 28K (1:500, mouse monoclonal antibody, Sigma C9848). Following primary incubation, sections were washed four times for 10 minutes at room temperature, then incubated with species-specific secondary antibodies, conjugated to Alexa Fluor 488 or 546 (1:250, in diluent solution described above), for 60 min at room temperature. Tissues were then washed four times in 1× PBS and mounted with Prolong Antifade solution with or without DAPI (Invitrogen).