Alexa fluor 488 conjugated anti mouse and alexa fluor 555 conjugated anti rabbit antibodies

To examine the effect of dasatinib on LPS-induced neuroinflammatory responses, BV2 microglial cells and mouse primary astrocytes were fixed with 4% paraformaldehyde for 10 min, washed three times with 1x PBS, and then incubated with anti-CD11b and anti-COX-2, anti-CD11b and anti-p-AKT, anti-CD11b and anti-p-ERK, or anti-p-STAT3 antibodies in GDB buffer (0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate, pH 7.4, and 450 mM NaCl) overnight at 4 °C. The next day, BV2 microglial cells and mouse primary astrocytes were washed three times with 1x PBS and incubated with Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit antibodies (1:200, Molecular Probes, USA) for 1 h at room temperature. The cells were mounted in DAPI-containing solution (Vector Laboratories, CA, USA), and images were captured from a single plane using a confocal microscope (Nikon, Japan) and analyzed using ImageJ software. Samples were analyzed in a blinded manner using 5–10 individual images.

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.