Anti rabbit alexa594

Immunocytochemistry (ICC) was used to detect the expression of M1 and M2 markers in BV2 microglia.^{42,43 (link)} BV2 cells were grown on 24-well plate with a glass slide chamber for overnight and were treated with either siTLR4/N3 or siCtrl/N3 complexes for 4 h, followed by OGD treatment. Cells were cultured for 24 h under normal cell culture conditions and fixed with 4% PFA in 0.1 M PBS. The fixed cells were rinsed using PBS, permeabilized with 0.1% Triton-X 100 for 10 min, and then incubated with 2% BSA in PBS for 1 h to block nonspecific binding. Then the cells were probed with primary antibodies for the M1 phenotype (iNOS, 1 : 400; Cell Signaling Technology, MA, USA) or the M2 phenotype (CD206, 1 : 400; Cell Signaling Technology, MA, USA). For visualization, the secondary antibodies, anti-rabbit Alexa-594 (1 : 1000; Cell Signaling Technology, MA, USA), or goat anti-rabbit IgG-FITC (1 : 1000; Abcam, Cambridge, MA) were used along with the nuclear marker DAPI (1 : 1000; Thermo Scientific, MA, USA). Images were obtained by a Nikon fluorescence confocal microscope (Nikon, C2⁺).