Evos fl auto 2 fluorescent microscope

Brain sections, 3 per mouse, containing the hippocampus were washed 3 times with PBS, followed by blocking in 5% normal donkey serum (NDS)/5% bovine serum albumin (BSA)/0.1% Triton/PBS solution for 1 h at room temperature while shaking. Sections were incubated overnight at 4 °C in 5% NDS/5% BSA/0.1% Triton/PBS solution with the following dilutions of primary antibodies: rabbit anti-IBA1 (cat #: 019–19741, Wako Chemicals, Richmond, VA, USA, dilution 1:2000), and goat anti-GFAP (cat #: ab53554, Abcam, Cambridge, MA, USA, dilution 1:2000). Next, sections were washed 3 times with PBS followed by incubation in 5% NDS/0.1% Triton/PBS with the following dilutions of secondary fluorescent antibodies: donkey anti-goat Alexa Fluor 488 (cat #: A-11055, Invitrogen, Carlsbad, CA, USA, dilution 1:1000), and donkey anti-rabbit Alexa Fluor 647 (cat #: A-31573, Invitrogen, dilution 1:2000). An additional 3 PBS washes were performed followed by mounting onto glass slides and coverslipping with Fluoromount G + DAPI solution (cat #: 0100–20, SouthernBiotech, Birmingham, AL, USA). Using an EVOS FL Auto 2 fluorescent microscope with an LED light source (Invitrogen), 20x images containing DAPI, GFAP, and IBA1 signals in the dentate gyrus (DG), CA3, and CA1 regions of the hippocampus of each hemisphere were acquired.