Immunohistochemistry

Tissue sections were retrieved from −20°C freezer and dried for 1 hour at room temperature, followed by antigen retrieval using R&D Systems Protocol (R&D Systems, Minneapolis, Minnesota). Immediately after antigen retrieval, slide edges were dried and a hydrophobic pen was used to surround the tissue sections (Newcomer Supply, Middleton, Wisconsin). A blocking/permeabilization step was conducted for 1 hour at room temperature with a solution of 5% Normal Horse Serum (Vector Laboratories, Burlingame, California), 0.2% Triton X-100 (Amresco, Solon, Ohio), diluted in PBS (primary and secondary antibodies were diluted in this solution as well). Tissue sections were then treated with primary antibody overnight at 4°C with the following antibodies: Neuronal marker anti-NeuN at 1:200 (EMD-Millipore, Temecula, California), astrocyte marker anti-GFAP at 1:400 (Dako, Carpinteria, California), oligodendrocyte lineage marker anti-Olig-2 at 1:200 (EMD-Millipore, Temecula, California), anti-HA tag at 1:200 (Sigma-Aldrich, St. Louis, Missouri), anti-Phospho-S6 Ribosomal Protein (Ser235/236) at 1:50 (Cell Signaling Technology, Danvers, Massachusetts), anti-wheat germ agglutinin (WGA) at 1:50 (Vector Laboratories, Burlingame, California). On the following morning, samples were washed 3× in PBS, and secondary antibody solutions were added for 1 hour at room temperature: donkey anti-rabbit IgG H&L (Alexa Fluor 647) at 1:200 (Abcam, Cambridge, Massachusetts), donkey anti-mouse IgG H&L (Alexa Fluor 488) at 1:200 (Abcam, Cambridge, Massachusetts), Rhodamine (TRITC) AffiniPure donkey anti-goat IgG (H+L) at 1:200 (Jackson ImmunoResearch, West Grove, Pennsylvania). Following secondary antibody treatment, samples were washed 3× in PBS. 2–3 drops of FluorSave reagent (Calbiochem, San Diego, California) was added to tissue sections, and coverslips (Fisher Scientific, Pittsburgh, Pennsylvania) were added. Slides were left to dry for 30 minutes at room temperature in the dark and then kept at 4°C.