Visualizing WGA-HRP in Brain Sections

Brains were frozen, and sectioned at 50 μm in the frontal plane using a sliding microtome (Model 860; American Optical Company, Buffalo, NY). Sections were stored in 0.1 M, pH 7.2 PB. In order to visualize the presence of WGA-HRP, sections were reacted using a tetramethylbenzidine (TMB) protocol that has previously been described (Bohlen et al., 2017 (link)). Briefly, sections were incubated in 0.25% ammonium molybdate (Fisher Chemical), 0.005% TMB (Fisher Chemical), and 2.5% EtOH in pH 6.0, 0.1 M PB. Following an incubation period, the reaction was initiated by adding 0.0125% H₂O₂ to the aforementioned solution and reacted for up to 18 hr at 4°C. The blue TMB reaction product was then stabilized by incubating sections in 5.0% ammonium molybdate in pH 6.0, 0.1 M PB. Sections were rinsed and mounted onto gelatinized glass slides and allowed to dry overnight. Then, they were counterstained with cresyl violet, dehydrated, cleared, and coverslipped.