Vectastain hardset mounting medium with dapi

Brain tissue was immunohistochemically processed in a Sequenza slide rack (Ted Pella Inc., Redding, CA) with all 4 experimental conditions represented in each batch. Slides were allowed to warm to room temperature for 20–30 min before rehydrating in 0.1 M PBS 3 × 5 min. Slides were then incubated in blocking solution (5% normal donkey serum in 0.1 M PBS + 1% bovine serum albumin [BSA]) for 30 min at room temperature. Primary antibodies (1:1000; goat anti-GFAP, Abcam ab53554, Abcam Inc., Cambridge, MA; rabbit anti-Iba1, Code No. 019-19741 lot# WEK6254, Wako Chemicals USA, Inc., Richmond, VA) were applied directly thereafter and incubated overnight at 4 °C. The following day, slides were washed 6 × 5 min in PBS then incubated with secondary antibodies (1:400; Alexa Fluor 488 donkey anti-rabbit, Code No. 711-545-152; Alexa Fluor 594 donkey anti-goat, Code No. 705-585-147; Jackson ImmunoResearch, West Grove, PA) at room temperature for 30 min. Slides were again washed 6 × 5 min before coverslipped with Vectastain Hardset Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Slides were stored in light-tight slide boxes at 4 °C.