Anti iba1 antibody

Brain sections were processed for immunofluorescence staining as previously described [46 (link)] and reported below. In brief, all sections were incubated overnight (O/N) in a humidified chamber at 37 °C using the following primary antibodies: anti-GFAP antibody (1:100; sc-33673; Santa Cruz Biotechnology), anti-IBA1 antibody (1:100; sc-32725; Santa Cruz Biotechnology). Sections were washed with PBS solution and incubated with IgG (H + L) highly cross-adsorbed goat anti-mouse secondary antibody, Alexa Fluor™ (1:1000 in PBS v/v, Molecular Probes, Altrincham, UK) for 1 h at 37 °C. After washing in PBS, nuclear staining with 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) (2 µg/mL) in PBS was added. Slides were observed and photographed at 40× magnifications using a Leica DM2000 microscope (Leica QWin V3, Cambridge, UK).

Immunostaining analysis of brain slices was performed according to the method of Duncan and Miller^{49 (link)} and Jang et al.^{22 (link)}. Microglial cells were visualized by staining with anti-Iba1 antibody (1:200, Santa Cruz). Apoptotic neuron cells were stained with anti-caspase-3 and anti-NeuN antibodies (1:500, Millipore). LPS were stained with ant-LPS antibody (1:200, Abcam). Briefly, the brains were cryoprotected in 30% sucrose-PBS and then frozen with optimal cutting temperature compound and stored at −80 °C until processed. Brain tissue blocks were cryosectioned at a thickness of 30 μm, stored at 4 °C in the storing solution (30% ethylene glycol in PBS), permeabilized in 0.5% Triton X-100 for 5 min, blocked in 10% bovine serum with tween 20-contained PBS for 30 min, and incubated for 16 h at 4 °C with antibodies. Secondary antibodies conjugated with Alexa Fluor 488 (1:1,000, Invitrogen) or Alexa Fluor 594 (1:500, Abcam) were then treated to visualize. Nuclei were stained with DAPI. Immunostained samples were scanned with a confocal laser microscope.