Quantitative Immunohistochemical Analysis of Alzheimer's Pathology

Primary antibodies used are summarized in Table 1. Immunostaining was performed as reported previously by our group [13 (link)–15 (link)]. Briefly, serial coronal sections were mounted on 3-aminopropyl triethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles. The sections for testing Aβ (4G8 antibody) were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H₂O₂ in methanol. The sections for testing total tau (HT7 antibody), and phospho-tau epitopes, were deparaffinized, hydrated, subsequently pretreated with 3% H₂O₂ in methanol, and then treated with citrate (10 mM) or IHC-Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum before incubation with primary antibody overnight at 4 °C. Next, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin-biotin complex method (Vector Laboratories) with 3,3′-diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by the immunoreactivities by using the software Image-Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD).