Avidin horseradish peroxidase hrp

The cortex and hippocampus of each mouse brain fixed with 3.7% formaldehyde. All specimens were embedded in paraffin and sliced into 4-μm thick sections. Sections were deparaffinized and then rehydrated, antigens were retrieved, and endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS. After the sections had been blocked with an IHC blocking reagent (Background Sniper; Biocare Medical, Concord, CA, USA) for 1 h, they were incubated with anti-Iba1 rabbit anti-mouse antibody (1:800 dilution, Biocare Medical, LLC, USA) or anti-cleaved caspase 3 rabbit anti-mouse antibody (1:500 dilution) (Cell Signaling Technology, Inc., Beverly, MA) in blocking reagent at 4°C overnight. Slides were then washed in PBS, incubated with species-specific biotinylated secondary antibody (1:200) for 30 min, washed with PBS again, amplified consecutively with avidin-horseradish peroxidase (HRP) (Vector Laboratories, Burlingame, CA, USA) and visualized by incubating them with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA). All slides were counterstained with hematoxylin (Mayer’s; ThermoShandon, Pittsburgh, PA, USA), dehydrated, and mounted. For negative controls, the procedure omitted the primary antibody.

Twenty-four hours after LPS administration, the mice were briefly anesthetized with isoflurane and then killed using cervical dislocation. The cortex and hippocampus of each mouse brain were fixed with 3.7% formaldehyde. All specimens were embedded in paraffin and sliced into 4 μm thick sections. Sections were deparaffinized and then rehydrated, antigens were retrieved, and endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS. After the sections had been blocked with an IHC blocking reagent (Background Sniper; Biocare Medical, Concord, CA, USA) for 1 h, they were incubated with anti-Iba1 (1 : 800 dilution) (Biocare Medical), anti-HDAC2 (1 : 200 dilution) or anti-HDAC5 (1 : 200 dilution) (Abcam, Cambridge, MA) rabbit anti-mouse antibodies in blocking reagent at 4°C overnight. Slides were then washed in PBS, incubated with species-specific biotinylated secondary antibody (1 : 200) for 30 min, washed with PBS again, amplified consecutively with Avidin horseradish peroxidase (HRP) (Vector Laboratories, Burlingame, CA, USA), and visualized by incubating them with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA). All slides were counterstained with hematoxylin (Mayer's; Thermo Shandon, Pittsburgh, PA, USA), dehydrated, and mounted. For negative Controls, the procedure omitted the primary antibody.