Immunohistochemical Profiling of Neurodegeneration

To detect CHOP expression, free floating sections including hippocampal area were stained as previously described^{56 (link)} using GADD153 (1:300, Novus Biological NBP213172) antibody followed by Alexa Fluor Goat α-Rabbit IgG-594 secondary antibody. Tissue was mounted onto slides and coverslipped with Prolong Gold Antifade reagent. ATF4 staining was performed using the primary ATF4/D4B8 (1:000, Cell Signaling 11815S) and anti-rabbit secondary (1:5000, Southern Biotech) antibodies. To prepare tissue for counting neuronal density, sections of 50 μm thickness were incubated with biotinylated NeuN antibody (1:3000, EMD Millipore MAB377B), mounted, and counterstained with 0.05% cresyl violet (Nissl) followed by dehydration with Xylenes and coverslipped with DPX solution (Sigma-Aldrich 06522). The Gallyas silver staining was used to detect tau positive neurofibrillary tangles as we have done previously^{58 (link)}. Animals with low or poor expression of the virus in the hippocampal region were excluded in the analysis.