Goat anti iba1 monoclonal primary antibody

BV2 cells were cultured on 24-well plates and transfected as described above. 24 h post-transfection, cells were stimulated with 1000 ng/ml LPS for 12 h, followed by collection of bright-field images using a Nikon microscope. The cells were then rinsed with PBS, fixed in 4% paraformaldehyde in PBS at RT for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were subsequently blocked in 2% bovine serum albumin in PBS at RT for 1 h, followed by incubation with a goat anti-Iba1 monoclonal primary antibody (1:500, Abcam, United States) at 4°C overnight. The following day, the cells were washed 3 times with PBS, and incubated sequentially with a donkey anti-goat IgG secondary antibody Alexa 488 (1:1000, Abcam, United States) at RT for 2 h, the cytoskeleton red fluorescent probe ActinRed (1:50, KeyGEN BioTECH, China) at RT for 20 min, and DAPI at RT for 5 min, and finally washed 3 times with PBS. Fluorescence intensity was detected using a Zeiss LSM710 fluorescence microscope.

Brain samples from WT and Dcf1-KO mice (C57BL/6 male mice, 2–3 months-old) were cut by frozen sectioning. Slices were rinsed 3 times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 40 min. The slices were subsequently blocked in 5% bovine serum albumin (Invitrogen, United States) in PBS at RT for 2 h, followed by incubation with a goat anti-Iba1 monoclonal primary antibody (1:500, Abcam, United States) at 4°C overnight. The following day, the slices were washed 3 times with PBS, incubated sequentially with a donkey anti-goat IgG secondary antibody Alexa 488 (1:1000, Abcam, United States) at RT for 2 h and the nuclear stain DAPI (Invitrogen, United States) at RT for 10 min, and finally washed 3 times with PBS. Fluorescence intensity was detected using a Zeiss LSM710 fluorescence microscope. All animals were treated in accordance with the guidelines of the Society for Neuroscience Ethics Committee on Animal Research. The study design was approved by the Animal Ethics Committee of Shanghai University.