Anti f4 80 antibody

Most chemicals including 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) were obtained from Sigma Aldrich (St. Louis, MO). Radio-labeled glucose and oleate were purchased from PERKin-Elmer NEN (Waltham, MA). Anti-actin, SIRT1, and JNK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-SIRT3, SIRT5, Akt, phospho-Akt, AMPK, phospho-AMPK, ACC, phospho-ACC, phospho-JNK, insulin receptor, phospho-insulin receptor, acetylated lysine, PERK, phospho-PERK, Bip, p-38, phospho-p38, p65, phospho-p65, and IκBα antibodies were purchased from Cell Signaling Technology (Beverly, CA). Anti-F4/80 antibody was obtained from eBioscience (San Diego, CA). Anti-SIRT4 antibody was obtained from Abcam (Cambridge, UK).

Bone marrow-derived macrophage (BMDM) cells were isolated from C57BL/6 or BALB/c mice and induced with 20 ng/mL of GM-CSF (Peprotech, Rocky Hill, CT, USA) for 7 days. Meanwhile, the medium was replaced with fresh medium containing the cytokine and the adherent cells were harvested. Phagocytosis assays were performed by a co-culture of the BMDM macrophages with carboxyfluorescein succinimidyl ester-labeled (CFSE⁺) or GFP⁺ tumor cells at a ratio of 1:4 in a serum-free medium at 37 °C for 4 h, in low-attachment 96-well tissue culture plates (Corning, New York, NY, USA). The cells were harvested and the macrophages were identified by a flow cytometry using anti-F4/80 antibody (eBioscience, San Diego, CA, USA). Then, 7-AAD (eBioscience) was used to exclude the dead cells. The effects of azelnidipine (20 μM) on phagocytosis by BMDM cells were tested, and the anti-CD47 antibody (clone: miap301) were used as the positive control. Phagocytosis rate was determined with the formulation: F4/80⁺GFP⁺ or F4/80⁺CFSE⁺ cells/ Total F4/80⁺ cells.

Paraffin-embedded 5-µm kidney sections were deparaffinized and rehydrated. Endogenous peroxidase activity was first quenched by H₂O₂ peroxidase blocking reagent (DakoCytomation). Macrophages and neutrophils were detected by using an anti-F4/80 antibody (1:50; eBioscience) and an anti–Ly-6G antibody (1:50; BD Biosciences), respectively; for 30 minutes at room temperature (RT). Sections were then washed and incubated with a secondary biotinylated goat anti-rabbit antibody (1:500; Jackson Immunoresearch) for 30 minutes at RT. Streptavidin-HRP was added and coloration was revealed using diaminobenzidine (DAB) with the substrate chromogen system from Dakocytomation. The number of F4/80⁺ and Ly-6G⁺ cells was counted in 10 non-overlapping fields (x400 magnification). Nitrotyrosine staining was performed using an anti-nitrotyrosine antibody (1:400; Abcam) and the OptiView DAB IHC Detection Kit (Ventana Medical Systems) according to manufacturer’s instructions. Nitrotyrosine intensity in the renal cortex was quantified using NIH ImageJ software.

Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 20 min, and blocked with PBS containing 3% bovine serum albumin (BSA, Yeasen, China) for 1 h. Then, cells were incubated overnight with the following primary antibodies: anti-F4/80 antibody (eBioscience, USA), anti-iNOS antibody (Abcam, USA), anti-ARG1 antibody (Proteintech, China), anti-Collagen X antibody (Abcam), USA), and anti-Collagen II antibody (Abcam, USA). Alexa 488 or Alexa 594 dye-labeled secondary antibodies (Jackson, USA) were added and incubated for 1 h at room temperature. Nuclear staining was performed for 5 min by the addition of 4',6-diamidino-2-phenylindole (Beyotime, China) solution. Immunoreactivity was visualized using a fluorescence microscope (Carl Zeiss, Germany).

Cecal ligation and puncture (CLP) was performed as described previously^{53 (link)}. Briefly, female mice were anaesthetized using isoflurane, the abdominal skin shaved, and under aseptic conditions, a 1 cm midline incision performed. The cecum was ligated with a silk suture and punctured once through-and-through with a 19 gauge needle. The cecum was then gently squeezed to exteriorize a drop of feces and returned to the peritoneal cavity. The laparotomy site was closed with 4.0 silk and animals returned to their cages. After 24 h, clinical score was assessed as previously described^{54 (link)} and blood, peritoneal lavage fluid (PLF), liver and kidneys collected. Peritoneal lavage fluid was collected using 2 ml ice-cold phosphate-buffered saline (PBS) supplemented with EDTA (10 mM). Hemoglobin was measured in PLF after lysing red cells as previously described^{19 (link)}. Liver (right lobe) and one kidney were homogenized in ice-cold sterile PBS and the homogenates plated on lysogeny broth (LB) agar to determine the number of colony forming units (CFU). The other kidney was processed in paraffin for immunohistochemistry. Neutrophil count in the PLF was measured by flow cytometry using anti CD45, CD11b and Ly6G (clone1A8, eBiosciences) and macrophages analyzed using anti-F4/80 antibody (eBiosciences). Podoplanin expression was analyzed using antibody clone 8.1.1 (eBiosciences).

The skin sections were washed with PBS, and the samples were blocked in an antibody dilution buffer of 2% BSA/PBS for 15 min at room temperature (RT). After the blocking solution was removed, primary antibodies/markers were added to the antibody dilution buffer at 37 °C for 2 h: anti-CD3 (1:200) (eBioscience, San Diego, CA, USA) for T lymphocytes; anti-F4/80 antibody (1:200) (eBioscience) for macrophages. After washing with PBS, cells were incubated for 30 min at RT with secondary antibodies prepared at 1:500 in antibody dilution buffer: Alexa 594 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). After the secondary antibodies were removed and the tissues had again been washed with PBS, nuclear counterstaining was performed by incubating with 4’,6-diamidino-2-phenylindole (DAPI) solution (1 μg/mL in PBS; Fujifilm Wako Japan K.K.) for 10 min at RT. A mounting medium (ImmunoBioScience, Mukilteo, WA) was added to the sample slides before they were covered with cover slips and sealed with nail varnish. They were then evaluated under a fluorescence microscope (BZx-700, Keyence, Osaka, Japan). The positively stained cells in each sample were counted in five different high-power fields (×200).