Immunostaining Analysis of Brain Slices

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.