Rat anti cd16 32

Free-floating sections were washed with PBS (3 × 10 min) and incubated at 4 °C for 72 h with continuous rotation in medium containing 0.2% (v/v) Triton X-100 (Probus S.A.), 5% (v/v) normal donkey serum (Sigma-Aldrich), and the following primary antibodies diluted in PBS: rat anti CD16/32 (1:200; #553142, BD Biosciences-Pharmigen, Franklin Lakes, NJ, USA), goat anti-CD206 (1:200; #AF2535, R&D Systems, Minneapolis, MN, USA), rat anti-CD45 (1:1000; #MCA1388, Bio-Rad Laboratories, Hercules, CA, USA), and rabbit anti-Iba1 (1:1000; #019-19741, Wako Pure Chemical Industries, Osaka, Japan). Then, sections were incubated with the appropriate Cy2- or Cy3-conjugated secondary antibodies (1:500; Jackson ImmunoResearch, West Grove, PA, USA) in PBS for 2 h at room temperature and counterstained with DAPI (4’,6-diamidino-2-phenylindole; 1:10,000; Sigma-Aldrich) to identify cell nuclei. Sections were washed with PBS, mounted on gelatin-coated microscope slides (VRW), and covered with fresh anti-fading mounting medium (70% v/v glycerol, 5% w/v n-propyl gallate, 0.42% w/v glycine, 0.03% w/v azide). For all different combinations of antibodies used in double-immunofluorescence experiments (Iba1/CD16/32; Iba1/CD206 and Iba1/CD45), two sagittal sections of the cerebellar vermis (distance: 180 µm) of each mouse were evaluated.

On the 7th day, the sciatic nerves were removed, fixed with 4% paraformaldehyde overnight, and then dehydrated sequentially with 15% and 30% sucrose. When the tissue sank to the bottom, nerves were embedded in Tissue‐tek medium (SAKURA) and snap‐frozen in liquid nitrogen to expose antigenic sites for staining. Transverse sections (8 μm) were prepared on a cryostat (Leica Microsystems LM3050S). Fluorescence immunohistochemistry of the frozen sections was performed using standard protocols provided by the antibody manufacturers. Primary antibodies included the following: goat anti‐CD206 (1:200; R&D Systems), rat anti‐CD16/32 (1:500; BD), and rabbit anti‐Iba‐1 (1:500; Wako). Images were taken using a fluorescence microscope (Olympus PX51) and analyzed with Adobe Photoshop 5.0 software. Data are expressed as the percentage of Iba‐1+ cells also positive for CD16/32 or CD206.

BV2 microglia were fixed in 4% paraformaldehyde for 15 min. After rinses in PBS, the cells were incubated with the following primary antibody: rat anti-CD16/32 (BD Biosciences Pharmingen, CA, USA) and goat anti-CD206 (R&D Systems, MN, USA) overnight at 4 °C, followed by secondary antibodies for 1 h with extensive washing. Images were captured by Nikon Optical TE2000-S inverted fluorescence microscope.

Single cell suspensions acquired from spleens and blood were first blocked with rat anti-CD16/32 (1:50, BD biosciences, Cat# 553,141) for 15 min at 4 ℃ and then incubated with CD4-APC (1:100, Biolegend, Cat# 100411) for 30 min at 4 ℃. Data were acquired on CytoFLEX3 (Beckman Coulter, USA) and analyzed with Flowjo V10 (TreeStar, Ashland, OR, USA).

Forty-eight hours after SAH, mice were anesthetized and perfused transcardially with phosphate-buffered solution (PBS) and with 4% paraformaldehyde. The brains were rapidly isolated and post-fixed in 4% paraformaldehyde for 4 h, followed by overnight immersion in phosphate buffer containing 30% sucrose. The brains were then embedded in OCT solution, and 8-μm coronal brain cryosections were prepared. Sections were fixed in ice-cold acetone and blocked with 10% goat serum for 2 h. Next, the sections were incubated at 4°C overnight with the following primary antibodies: rat anti-CD16/32 (1:150, BD Pharmingen, San Diego, CA, USA), rabbit anti-Peli1 (1:250, Abcam, Cambridge, UK). After being washed in PBS, the sections were incubated with appropriate secondary antibody for 30 min at 37°C: TRITC-conjugated goat anti-rat IgG (1:200, Proteintech, Wuhan, Hubei, China) and DyLight 488-conjugated goat anti-rabbit IgG (1:200, Abbkine, California, USA). The nuclei were stained with DAPI. The stained sections were visualized with a fluorescence microscope (Nikon, Tokyo, Japan).

Brain slices were obtained and used for immunofluorescence staining, as previously described (Ma et al. 2016 (link)). The primary antibodies were goat anti-CD206 (1:200; R&D Systems, Minneapolis, MN, USA), rat anti-CD16/32 (1:100; BD Pharmingen, San Jose, CA, USA) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:200; Wako Pure Chemical Industries, Osaka, Japan). The fluorophore-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA) used were donkey anti-goat antibody conjugated to Alexa 488 (1:200), donkey anti-rat antibody conjugated to Alexa 594 (1:200) and goat anti-rabbit antibody conjugated to Alexa 594 (1:200). Images were captured using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Mean merged cell counts were calculated from three randomly selected microscope fields in the cortex of each section, and three consecutive sections were analyzed from each brain. All images were manually counted. Data are expressed as mean number of cells/mm². The person evaluating TUNEL staining and immunofluorescence staining was blinded to the treatment group of the mice.