Goat anti iba1 antibody

Immunofluorescence was performed as previously described by our laboratory [22 (link)]. Briefly, tissue sections were fixed in 4% polyformaldehyde for 1 min, washed three times with phosphate-buffered saline (PBS), permeabilized with 0.4% Triton X-100 for 30 min, and blocked with goat serum working liquid (Wuhan Boster Biological Technology, Wuhan, China) for 1 h. The sections were then incubated overnight with mixed primary antibodies at 4 °C, washed in PBS to remove unbound primary antibodies, and incubated with secondary antibodies in the dark at RT for 1 h. The primary antibodies included rabbit anti-GPR120 (1:100; Affinity, China), mouse anti-NeuN antibody (1:100; Abcam, USA), mouse anti-GFAP antibody (1:100; SAB, China), and goat anti-Iba1 antibody (1:100; Abcam, USA). The fluorophore-conjugated secondary antibodies used were goat anti-mouse Alexa Fluor 650 (1:100; Abcam, USA), goat anti-rabbit Alexa Fluor 488 (1:100; Wuhan Boster Biological Technology, China), donkey anti-goat Alexa Fluor 549 (1:100; Wuhan Boster Biological Technology, China), and donkey anti-rabbit Alexa Fluor 488 (1:100; Wuhan Boster Biological Technology, China). Images were captured by confocal laser scanning microscopy (Leica, Wetzlar, Germany). The fluorescence intensity was analyzed using Image-Pro Plus 6.0, and colocalization analyses were performed using ZEISS.

Senile plaques were stained with 0.01% BSB as previously described [3 (link),36 (link)]. To analyze the activation of plaque associated astrocyte and microglia, BSB-stained brain sections were further incubated with primary antibodies (i.e., mouse anti-GFAP antibody (1:500, Millipore, Temecula, CA, USA) or goat anti-Iba-1 antibody (1:300, abcam)) at 4 °C overnight; washed with PBS; incubated with fluorescein isothiocyanate-donkey anti-mouse IgG and Alexa Fluor 647-donkey anti-goat IgG (1:300, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. Slice was mounted with Aqua Poly/Mount (Polysciences, Inc., Warrington, PA, USA). Fluorescent images of BSB staining were taken using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy, Jena, Germany).

For immunofluorescence staining, brain sections were washed with phosphate‐buffered saline containing 1% Triton for 3 times and then incubated with goat anti‐IBA1 antibody (1:300, Abcam), rabbit anti‐iNOS antibody (1:100, Genetex), or rabbit anti‐Arg1 antibody (1:200, Genetex) for 24 h at 4°C. After being washed with PBS for 3 times, the sections were incubated with CY3‐labeled goat anti‐rabbit and FITC‐labeled goat anti‐rat secondary antibodies (1:1000 for both, Millipore) at room temperature for 2 h. Lastly, sections was incubated with the nuclei marker DAPI (1 ng/u, Sigma) for 5 min at room temperature. Fluorescent signals were detected using a confocal fluorescence microscope (Flv100i, Olympus).