Goat anti mouse igg h l cy3 preadsorbed

CCRF-CEM cells were treated with 30 µM of compound (1) for 24 h. Then, the cells were washed with PBS and fixed with 3.7% paraformaldehyde for 30 min at room temperature. The cells were blocked for 1 h at room temperature using a blocking buffer (5% FBS and 0.3% Triton X-100 in PBS). Primary antibodies [rabbit α-tubulin antibody (Abcam, Cambridge, UK) and mouse PLK1 monoclonal antibody (Thermo Fisher Scientific)] were added and allowed to stand for 2 h at room temperature (dilution 1:1000). Then, the cells were washed with PBS three times. Secondary antibodies [goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (Abcam) or goat anti-mouse IgG H&L (Cy3^®) preadsorbed (Abcam)] were subsequently added (dilution 1:1000). Then, the cells were washed with PBS, stained using 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich), and mounted using Fluoromount-G^® (Southern Biotech, Birmingham, AL, USA). Fluorescence imaging was performed using an EVOS SL digital inverted microscope (Life Technologies).

The purification of the astrocytes was confirmed by glial fibrillary acidic protein immunocytochemical staining, and the purification of the microglia was confirmed by ionized calcium-binding adapter molecule 1 immunocytochemical staining. Cultured astrocytes and microglia were first fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 minutes. Bovine serum albumin (5%; Invitrogen) was used as a blocking agent at room temperature for 1 hour. The cells were then incubated with anti-glial fibrillary acidic protein antibody (Cat# ab7260; mouse, 1:5000, Abcam, Cambridge, MA, USA) or anti-ionized calcium-binding adapter molecule 1 antibody (Cat# ab48004; mouse, 1:1000, Abcam) at 4°C for 12–16 hours. The secondary antibodies used were goat anti-mouse IgG H&L (Cy3®) preadsorbed (Cat# ab97035; 1:500, Abcam). Hoechst 33342 (Abcam) was used to label the cell nucleus. The reactions were visualized under a TCS SP2 confocal microscope or a ZEISS-AX10 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The morphology of the microglia and astrocytes was examined using ImageJ (Rawak Software Inc., Stuttgart, Germany).