Fitc anti mouse f4 80 antibody

The paraffin sections of right lung lobes were deparaffinized. After inhibiting endogenous peroxidase activity and repairing antigen, the non-specific binding sites were blocked with 10% bovine serum albumin (BSA) and incubated with primary antibodies including rabbit anti-NE (bs-23549R, Bioss, Beijing, China) and rabbit anti-CitH3 (AF0863, Affinity, Colorado, United States) for 12 h at 4°C and washed with PBST, then staining tissues with Goat Anti-Rabbit IgG H&L (Alexa Fluor®488, ab150077, Abcam, London, UK) and Goat Anti-Rabbit IgG H&L (Alexa Fluor®647, ab150115, Abcam) secondary antibodies, respectively. The expression levels of NE and CitH3 were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, United States) after being detected by an optical microscope (Vectra 3, PerkinElmer, Waltham, United States) (Liu et al., 2022 (link)).
In addition, dewaxed and blocked tissues were incubated with FITC anti-mouse F4/80 antibody (1:50, 123107, Biolegend, California, United States), PE anti-mouse Ly-6G antibody (1:50, 127607, Biolegend) and FITC anti-mouse MPO antibody (1:50, ab90812, Abcam) for 1 h at 37°C, respectively, followed by staining with DAPI (1:200) to label cell nuclei for 10 min at 37°C. The expressions of these genes were detected by optical microscope and quantified using ImageJ software (National Institutes of Health).

We harvested peritoneal cavity cells 24 hours after CLP. The cells were stained with a PE anti-mouse/human CD11b antibody (101207, BioLegend, Inc., San Diego, California, USA) and FITC anti-mouse F4/80 antibody (123108, BioLegend, Inc.) or PE Rat IgG2b, κ isotype ctrl antibody (400608, BioLegend, Inc.) and FITC Rat IgG2a, κ isotype ctrl antibody (400505, BioLegend, Inc.) for 30 minutes at 4°C after blocking the nonspecific Fc receptor using the Fc blocking reagent (130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes at room temperature. After staining, the cells were washed immediately and resuspended in AutoMACS Runnig Buffer (130-091-221, Miltenyi Biotec). Fluorescence data were collected using a SH800 (Sony Biotechnology Inc., Tokyo, Japan). The flow cytometry files were analyzed using FlowJo software (Ver. 10.8.1, Becton, Dickinson and Company).

The peritoneal macrophages isolated from Mac IKKα−/− mice or WT mice were washed twice with PBS and centrifuged at 1000 rpm for 5 minutes. Approximately 1 × 10⁶ cells were stained for 30 minutes at room temperature with PE anti-mouse CD14 antibody (123309; Biolegend) and FITC anti-mouse F4/80 antibody (123107; Biolegend) for detecting macrophages, Alexa Fluor® 488 anti-mouse CD206 antibody (141709; Biolegend), and PE anti-mouse F4/80 antibody (123109; Biolegend) for detecting M2 macrophages. Then, the stained cells were washed twice, resuspended in FACS buffer, and analyzed on a Beckman FC 500 Flow Cytometer with FlowJo software.

For flow cytometry analysis of in vitro mouse immune cells, the mouse T cells were incubated with 1× Golgiplug (BD Biosciences) for 6 hours. Immune cells were stained with surface antibodies in PBS for 30 minutes as described previously (51 (link)). Fixation and permeabilization processes were carried out with fixation buffer (BD Biosciences) according to the manufacturer’s protocols. The immune cells were then stained with antibodies in PBS to detect proteins. The following antibodies were used for staining: Brilliant Violet 510 anti-mouse/human CD11b (BioLegend, catalog 101245), FITC anti-mouse F4/80 antibody (BioLegend, catalog 123107), PerCP/Cyanine5.5 anti-mouse CD4 antibody (BioLegend, catalog 100540), APC anti-mouse CD206 antibody (BioLegend, catalog 141708), Brilliant Violet 421 anti-mouse CD86 antibody (BioLegend, catalog 105032), and PE anti-mouse FoxP3 antibody (Elabscience, catalog E-AB-F1238D). The flow cytometry was run using FACSCelesta flow cytometer (BD Biosciences), and the results were analyzed with FlowJo V10.7.1.

The femur and tibia were removed from eight-week-old C57BL/6 mice, and single-cell suspensions were prepared in Hank’s Balanced Salt Solution (PB180323, Procell) containing 5% FBS. Red blood cells were lysed using red blood cell lysis buffer. The samples were centrifuged, and the supernatant was discarded. The cells were resuspended and cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (CB34, Novoprotein) [20 (link)]. BMDMs were incubated with APC anti-mouse/human CD11b antibody (101,212, Biolegend) and FITC anti-mouse F4/80 antibody (123,108, Biolegend), identified by flow cytometry.