Biotinylated goat anti mouse secondary antibody

Immunohistochemistry for p16 was performed using a commercial mouse anti-human monoclonal antibody (BD Biosciences, San Jose, CA, USA) whose specificity for canine p16 had been previously validated [21 (link),26 (link)].
Endogenous peroxidase activity was blocked by incubation for 15 minutes with 1% hydrogen peroxide in methanol. Antigen retrieval was obtained by incubation in EDTA buffer (pH 9.0) for 20 minutes at 95°C, with a 20-minutes cooldown. Slides were then incubated for 60 minutes with p16 antibody at a dilution of 1:100. Sites of primary antibody binding were identified by incubation with biotinylated goat anti-mouse secondary antibody (Dako, Glostrup, Denmark) at a dilution of 1:200 in blocking solution. Sections were incubated with a commercial streptavidin–biotin–peroxidase kit (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, California, USA) for 30 minutes; 3,3′-diaminobenzidine was used as chromogen. Sections were counterstained with Papanicolaou’s hematoxylin.
Epithelial cells within the basal layer of epidermis or mucosal surfaces often exhibited weak immunoreactivity and were used as an internal positive control. Negative controls were obtained by omitting the primary antibody.

Nine free floating tissue sections comprising the hippocampal formation of three animals per group were processed for immunohistochemistry. Brain sections were washed (3 × 10 min) with a solution buffer containing PBS 0.125 M (pH 7.4), 0.5% Triton X-100 and 0.1% BSA. After washing, sections were treated with methanol and H₂O₂ to inhibit endogenous peroxidase activity and incubated in 70% formic acid for 7 min to expose the epitope. Sections were incubated overnight with primary antibody 4G8 (anti 18–22 Aβ amino acids; 1:500, Chemicon International, Billerica, MA, USA) or anti-pTau primary antibody AT8 (1:1000, Thermo Fisher Scientific, Rockford, USA) at 4 °C. After washing, sections were incubated sequentially with biotinylated goat anti-mouse secondary antibody (1:500, DakoCytomation, Glostrup, Denmark) for 2 h, an ABC kit immunoassay detection system (Vector Labs, Burlingame, CA, USA) for 90 min, and developed with 3,3′-diaminobenzidine (DAB) solution (Peroxidase substrate kit, Vector Labs, Burlingame, CA, USA). Sections were then washed in distilled water before dehydrating and mounting in DPX (VWR-BDH Dublin, Ireland).
Six different areas covering the entire hippocampus from two sections on anatomically comparable planes/animal were analysed. 4G8 labeling, divided into major and minor plaques, and AT8 intraneuronal immunoreactivity were blindly assessed.