Immunohistochemical Analysis of Crif1 in Tg6799 Mice

Immunocytochemical staining was performed as previously described.^{55 (link)} Images were obtained using a confocal laser scanning microscope (FV10i-w, Olympus). For histological analyses, Tg6799 mice were killed at 6 months of age (n=6 per group), brains were sliced and were stained for Crif1 (Santa Cruz Biotechnology; 1 : 250).^{40 (link)} Immunohistochemistry on human brain tissues was performed as previously described.^{59 (link)} Briefly, postmortem tissue blocks were cut into 40 μm-serial sections on a freezing sliding microtome (Model 860; American Optical Company, Buffalo, NY, USA). Sections were stored in cryoprotectant solution (30% glycerol, 30% ethylene glycol, 0.1% sodiumazide in PB; pH 7.4) at −20 °C. Tissue sections were incubated for 48 h with anti-Crif1 (Santa Cruz Biotechnology; 1 : 100), then the sections were incubated in biotinylated horse anti-mouse IgG antibody (Vector Laboratories, Burlingame, CA, USA; 1 : 500) for 2 h at room temperature. Subsequently, the sections were incubated with streptavidin (Invitrogen; 1 : 5000) for 2 h at room temperature. Nickel-enhanced DAB/peroxidase reaction (0.02% DAB (Sigma-Aldrich), 0.08% nickel-sulfate, 0.006% hydrogen peroxide) was used to visualize the reaction product.