Anti iba1 primary antibody

BV2 cells were mock-treated, infected with ZIKV_PE243 or ZIKV_MR766, with an MOI of 1, or stimulated with LPS + ATP as a positive control. After 24, 48, and 72 hpi, cells were fixed and permeabilized as described previously. Then, the cells were incubated overnight with the primary anti-Iba1 antibody at 1:500 (Wako, code 019-19741), anti-CD68 antibody at 1:500 (Bio-Rad, MC1957, Hercules, CA, USA), or with anti-MHC II conjugated with phycoerythrin—PE (eBioscience, San Diego, CA, USA, ref. 12-5322-81). The cells were then stained with the respective anti-IgG secondary antibodies conjugated to AlexaFluor488 or AlexaFluor647. To stain cell nuclei, a mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) (blue) (Vectashield—Vector Laboratories, Mowry Ave Newark, CA, USA) was used. Fluorescence microscopy was performed simultaneously with the same antibody solutions, and all micrographs were taken with the same settings using a Zeiss LSM 710 confocal microscope equipped with a 63× objective and digital imaging system (Roper Scientific camera and Zen (black edition) software).

The P30 mice were injected with 10 mL/kg (stock solution of 1 mg/mL) of lipopolysaccharide (LPS) intraperitoneally at P30, as previously described [32 (link)]. After 6 and 24 h, the mice were sacrificed, and their eyeballs were enucleated for Western blot and qRT-PCR analyses, respectively. At 24 h post-injection, immunostaining to identify Iba1-positive cells was also performed. For Iba1-positive microglial cell counting, the cryosections were rinsed with PBS and then blocked in 5% normal donkey serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. The retinal cryosections were then incubated in the primary anti-IBA-1 Antibody (Wako Chemicals, 019-19741, Osaka, Japan). The secondary Alexa Fluor 555 or 488 anti-rabbit antibodies (ThermoFisher Scientific, A31572; A21206, Eugene, OR, USA) diluted in PBS (1:500) were applied for 2 h at room temperature. Nuclei were visualized by counterstaining with DAPI in a fluorescence mounting medium (Southern Biotech, 0100-20, Birmingham, AL, USA), and images were acquired with a Keyence BZ-X800 Fluorescence Microscope (Itasca, IL, USA). Investigators blinded to the results counted the number of Iba1-positive cells in the retinal sections. Fluorescent images were taken, and the Iba1-positive cells were calculated in a vision field of 200 µm².