ab20874 staining Apolipoprotein E in rat brain tissue by Immunohistochemistry (Frozen sections). After fixation in 4% PFA in PBS (pH 7.4), brains were cryoprotected in 30% sucrose in PBS at +4°C, frozen, and thereafter kept at -80°C until used. For the immunostaining, brains were cryosectioned in 30 µm slices, collected in PBS (pH 7.4), and processed in a free-floating system. Slices were first incubated in the blocking solution (BS) containing 2% bovine serum albumin, 2% goat serum, and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature, and thereafter with the primary antibody for 24–48 hours at +4°C in BS at a 1/1000 dilution. After washing in PBS, slices were incubated with the biotin-conjugated secondary antibody (1/4000) in BS, rinsed with PBS, and incubated with the avidin-peroxidase conjugate in BS for 1 hour at room temperature. The staining was detected using 3,3-diaminobenzidinetetrahydrochloride as a chromogen.