Alexa fluor 488 or 555 conjugated secondary antibody

For immunostaining, the fixed brains were cut coronally at 50 μm thickness by a vibratome (Leica, Buffalo Grove, IL). Sections were permeabilized in 0.1% Triton X-100 in PBS for 10 min and incubated in blocking buffer (10% BSA in PBS) overnight at 4°C. Then, sections were incubated with the rabbit polyclonal anti-TMEM119 (1:200, 27585-1-AP, Proteintech), mouse monoclonal anti-PCNA (1:50, 60097-1-Ig, Proteintech), mouse monoclonal anti-IL-1β (1:200, sc-52012, Santa Cruz), rabbit monoclonal anti-Arg1 (1:500, 93668, Cell Signaling Technology), and Alexa Fluor 488 or 555-conjugated secondary antibodies (1:500, Cell Signaling Technology). Nuclei were counterstained with DAPI (1 μg/mL, Sigma). Fluorescence images were taken using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). For cell counting, six ROIs (the region of interest) along the border of the ischemic ipsilateral hemisphere were counted. Data were expressed as the mean value of the cells in the ROIs. Cell counting was performed by blinded investigators.

The cells (2.0 × 10⁵) were plated onto a 20 mm circle cover glass and treated as indicated. The cells were first fixed with 4% formaldehyde for 10 min, and permeabilized with 0.25% Triton X-100 for 15 min. The cells were then successively blocked in PBS with 3% BSA for 1 h, probed with primary antibodies (anti-Chk1 pS345, #2348, 1:50; anti-γH2AX, 05-636, 1:250) overnight at 4 °C, and incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies (1:400, Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. The cells were finally soaked in DAPI (Vector Laboratories, Burlingame, CA, USA), mounted on slides, sealed with nail polish, and dried in the dark. Images were acquired with a FLUOVIEW FV1000 (Olympus, Shinjuku, Japan) and quantified by ImageJ.