Rabbit anti cd11b

Primary and secondary Abs used for immunolabeling: mouse anti-Ki67 Ab (Dako), rat anti-BrdU (Abcam), mouse anti-Muc2 and rabbit anti-chromogranin A (Santa Cruz), rabbit anti–caspase-3 (Cell Signaling); rabbit anti–E-cadherin (BD Bioscience); rat or rabbit F4/80, rat anti-7/4 (Abcam); rat or rabbit anti-Ly6C, Ly6G, and Gr-1 (BD Pharmingen); rabbit anti-CD11b; and rat IgG and rabbit IgG (Abcam). Immmunolabeling was visualized using an appropriate combination of species-specific Alexa Fluor–conjugated secondary Abs (488, 568, and 647 nm) raised in mouse, donkey, or goat (Invitrogen).

Sections stained with immunofluorescence were incubated overnight in a cold room with the antimouse primary antibody (as required), rabbit anti-CD11b (1:500, Abcam), rabbit anti-CD4/80 (1:500, Abcam), anti-CD4 (1:500, Abcam), or rabbit anti-CD8 (1:200, Cell Signaling Technology, Danvers, MA, USA). The sections were subsequently immunohistochemically stained with specific fluorophore-conjugated goat anti-rabbit (F-2765, 1:500, Invitrogen). Fluorophore FITC (488 nm excitation wavelength), CY3 (555 nm excitation wavelength), or CY5 (670 nm excitation wavelength) antibody. Sections were then incubated with DAPI, and the slides were scanned (×40) using a Panoramic Midi Scanner (3DHISTECH, Budapest, Hungary). High-magnification images were obtained using Case Viewer software, and intensity of fluorescence was measured using Fiji/ImageJ software following standard protocol. Corrected total fluorescence (CTF) was analyzed following the standard formula [CTF = Integrated density − (Area of selected cell × Mean fluorescence of background readings)], and average intensity was calculated.

Medium was removed after incubation, and cells were fixed with 4% paraformaldehyde. The fixed cells were washed and permeabilized or not with 0.1% Triton X-100 (Sigma). After incubation with 5% bovine serum albumin (Sigma), cells were incubated with Rabbit anti-GFAP antibodies (1:1000, Abcam, Cat#ab7260), rabbit anti-Cx43(1:75, Cell Signaling Technology, Cat#3512) or Rabbit anti-CD11b (1:1000, Abcam, Cat#ab128797) at 4 °C overnight. Then cells were washed, and the secondary antibodies conjugated with Alexa Fluor-488 (1:500; Abcam, Cat#ab150077) were applied. For nucleus labeling, the cells were incubated with DAPI (1:1000; Beyotime, Cat#C1006). Observations were performed using a fluorescent microscope (Leica).

U937 cells plated on glass 25 mm² coverslips (2.5 × 10⁵ cells/well in a six-well plate) were differentiated with 25 ng/mL PMA for 48 h. Cells were washed and live stained with mouse anti-SerpinB2 (American Diagnostica, #3750) and fluorescein isothiocyanate (FITC)-labeled anti-mouse secondary antibody (Chemicon) at 4°C. Costaining for CD11b used rabbit anti-CD11b (Abcam) and anti-rabbit FITC (Chemicon), and anti-mouse Texas Red-X for detecting the anti-SerpinB2 antibody (Molecular Probes, Eugene, OR). Cells were washed at 4°C and fixed in 4% paraformaldehyde. Coverslips were mounted onto slides using ProLong Gold (Invitrogen, Carlsbad, CA) and analyzed on a Leica TCS confocal microscope (Leica Microsystems, North Ryde, NSW, Australia).

Rats were anesthetized with 2% sodium pentobarbital in saline (60 mg/kg, i.p.; Sigma, St Louise, MO, USA) and perfused with saline and then perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) on day 14 after surgery. Brains were collected and fixed in the 4% PFA for 2 h and dehydrated in 30% sucrose at 4°C overnight. 10-mm-thick coronally sections of the prefrontal cortex were cut and pasted on glass slides. After blocking with 10% norm goat serum for 1 h at room temperature, the slices were incubated with primary antibodies: rabbit anti-CD11b (1:250; Abcam, Cambridge, UK), rabbit anti-Iba1 (1:500; Wako, Japan), mouse anti-CD206 (1:500; Biolegend, San Diego, USA), or mouse anti-8-hydroxy-20-deoxyguanosine (8-OH-dG; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) in 1% BSA at 4°C overnight. After washing with PBS for 3 × 5 min, the slices were exposed to the secondary antibodies Alexa fluor 488/549 goat anti-rabbit and Alexa fluor 488/549 goat anti-mouse (1:400; Bioworld Technology, St. Louis Park, MN, USA), and DAPI (1:1,000; Sigma, St. Louis, MO, USA) for 1 h at room temperature. A confocal microscope (Leica, TCS SP2, Germany) was used to capture the fluorescent images. The immunofluorescence intensity was calculated by Image J (Wayne Rasband, National Institute of Health, USA).