Avidin biotin peroxidase complex abc kit

The immunoperoxidase method was chosen to perform immunohistochemical reactions at the light microscopic level, as we described in detail previously [64 (link)]. Hippocampal sections were incubated for one hour in 10% normal goat serum (NGS) diluted in 50 mM Tris buffer (pH 7.4) containing 0.9% NaCl (TBS) and 0.2% Triton X-100. After several washes in TBS, hippocampal sections were incubated in the primary antibody (anti-GABA_B1 or anti-β amyloid, both at a concentration of 1–2 µg/mL and diluted in TBS containing 1% NGS). Next, the sections were incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 in TBS containing 1% NGS, and then transferred into avidin–biotin–peroxidase complex (ABC kit, Vector Laboratories). Next, we revealed peroxidase enzyme activity using the chromogen 3,3´-diaminobenzidine tetrahydrochloride (DAB; 0.05% in TB, pH 7.4) and the substrate H₂O₂ (0.01% in distilled water). Hippocampal sections were then air-dried for few hours, mounted in DPX (Merk, Darmstadt, Germany), coverslipped and observed in a Leica photomicroscope (DM2000) equipped with differential interference contrast optics and a digital camera (DFC500).

Immunohistochemistry (IHC) and stereological analysis of F4/80+ cells in the dorsal hippocampus were performed as described previously [20 (link)]. Briefly, 9-month-old mice were perfused transcardially with PBS and immersion-fixed in 4% formaldehyde overnight. Following transfer to a sucrose gradient, 40 μm sections were cut using a cryostat. IHC was performed on free-floating sections using a monoclonal rat anti-mouse F4/80 primary antibody (1:10,000; Serotec, Raleigh, NC, USA) and a biotinylated goat anti-rat immunoglobulin G (IgG) secondary antibody (1:3000; Vector Laboratories, Inc., Burlingame, CA, USA), after which sections were treated with avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Inc., Burlington, CA, USA). Staining was visualized using diaminobenzidine (DAB; Vector Laboratories) and counterstained with hematoxylin (Fisher Scientific, Fair Lawn, NJ, USA). Stereological counting was performed using a Nikon 218912 light microscope interfaced with the StereoInvestigator software package (MicroBrightField, Williston, VT, USA). IgG leakage was assessed in adjacent sections (5 per mouse) as above except we used a biotinylated anti-mouse IgG antibody (1:3000).

CD68+ macrophages were detected in mouse tissues by immunohistochemistry using a CD68 (FA-11) rat monoclonal antibody (ThermoFisher Scientific, Rockford, IL, USA) at a dilution of 1:200. After deparaffinization, epitope retrieval was performed by immersing the sections in citrate buffer (Scytek, UT, USA), followed by heating in a pressure cooker. Thereafter, sections were successively exposed to solutions containing avidin and biotin to avoid false-positive staining reactions resulting from endogenous biotin, followed by casein and anti-CD68 incubation overnight. The day after, sections were exposed to the corresponding biotinylated secondary antibody (polyclonal rabbit anti-rat IgG) and then to the avidin-biotin-peroxidase complex (ABC kit), both from Vectorlab (Peterborough, UK). Presence of antigen in the sections was visualized by incubation with a chromogenic substrate mixture containing DAB and H₂O₂. Finally, sections were counterstained with luxol fast blue and mounted with a synthetic medium. The number of CD68+ macrophages was counted in 5 fields in each tumor tissue with an AxioCam MRC5 optical microscope at 400× magnification.