Immunocytochemistry of BV2 Microglia Cells

Immunocytochemistry of BV2 cells was conducted according to a previously methodology (6 (link), 7 (link)). In brief, cells were fixed for 10 min in 4% PFA, washed thrice with PBS, and then incubated with either anti-CD11b (Abcam, Cambridge, UK) and anti-p-STAT3^S727 (Abcam) or anti-CD11b and anti-p-NF-κB^S536 (Cell Signaling Technology, Danvers, MA, USA) antibodies overnight (Table 3). After washing the cells with PBS for 10 min, Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit antibodies (1:200, Molecular Probes, USA) were incubated for 1 h at room temperature. After washing thrice with PBS for 10 min, the cells were mounted with DAPI (Vector Laboratories, CA, USA), and fluorescence microscopy images were acquired (DMi8, Leica Microsystems, Wetzlar, Germany) and analyzed using ImageJ.