FFPE slides with mouse brain sections were deparaffinized and probed with primary antibodies as described before [11 (link)]. For antigen retrieval, slides receiving AT8 (1:1000; Invitrogen MN1020), AT100 (1:1000; Invitrogen MN1060), AT270 (1:1000; Invitrogen MN1050), AT180 (1:1000; Invitrogen MN1040) and glial fibrillary acidic protein (GFAP) (1:3000; Cell Signaling) antibodies were steamed in water at high pressure for 15 min, while Iba-1 (1:2000; Wako) antibody slides were steamed in citrate buffer pH 6.0 (Target Retrieval Solution, Dako). Slides were incubated in 3% hydrogen peroxide (Fisher Scientific) for 20 min to block endogenous peroxidase activity and then washed three times in PBS for 5 min each. Slides were then blocked in 2% fetal bovine serum (FBS) (Hyclone, GE) for 45 min before incubating in primary antibody diluted in block solution overnight at 4 °C. The following day, slides were washed and appropriate secondary antibody (ImmPRESS Polymer Reagent, Vector Labs) was applied for 30 min at room temperature. Following PBS washes, color was developed using 3,3’-diaminobenzidine (Vector DAB, Vector Labs) and slides were counterstained with haematoxylin (Vector Labs). Next, brain sections were dehydrated in a series of ethanol, cleared in xylene, mounted in Cytoseal-60 media (Fisher Scientific) and coverslipped.
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