Anti iba1

Cells were fixed for 10 min in 4 % paraformaldehyde, permeabilised and incubated with primary antibodies in 1× PBS with 10 % normal serum. Primary antibodies used were anti-Olig2 (1:500; Millipore), anti-GFAP (1:400; Millipore), anti-Ki67 (1:1,000; Abcam), anti-Nestin (1:1,000; Abcam), anti-Sox2 (1:200; Santa Cruz), anti-O4 (Sigma, 1:200), anti-Iba1 (1:200, Biocare), TuJ1 (1:1,000, Covance), anti-GFP (1:2,000, Abcam), anti-bromodeoxyuridine (BrdU) (1:250, Abcam) and anti-NFκB-p65 (1:500; Abcam). Primary antibodies were visualised using specific AlexaFluor secondary antibodies (Molecular Probes), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted in Prolong Gold anti-fade mounting medium (Molecular Probes) and analysed using Zeiss AxioImager Z1 microscopes and AxioVision software.

Cortices were isolated from P21 Swiss Webster and collected in calcium/magnesium-free Hank’s balanced salt solution (HBSS), trypsinised and DNAseI treated (50 μg/ml, Sigma) for 20 min at 37 °C and mechanically dissociated into a homogenous cell suspension [28 (link)]. Following washes and centrifugation, mixed glial cells were plated onto PDL (Sigma) in DMEM/F12 (Invitrogen) with 100 U/ml penicillin/100 mg/ml streptomycin (Sigma) and 10 % FBS (Biosera). Once confluent, cultures were shaken overnight at 180 rpm to remove microglia and oligodendrocytes and treated for 4–7 days with 20 μM cytosine arabinoside (AraC) to kill remaining dividing cells and obtain essentially pure astrocytes (>98 %), determined by immunofluorescence using anti-glial fibrillary acidic protein (GFAP) (Millipore) and anti-S100β (Dako) for astrocytes, anti-Iba1 (Biocare, microglia), anti-O4 (Sigma, oligodendrocytes) and TuJ1 (Covance, neurons).

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.