Pe anti mouse f4 80 antibody

Ba/F3 P210 BCR-ABL cells (5 × 10⁶ cells) were administered via intravenous (i.v.) injections into the lateral tail veins of immunosuppressed mice (n = 12), on days 11, 14, and 17 (Fig. 1), thereafter called leukemia mice (n = 12). To determine the presence of Ba/F3 BCR-ABL P210 cells in mice peripheral blood, we used the presence of both CD45 (a leukocyte marker, most expressed in lymphocytes) and F4/80 surface (https://cell.brc.riken.jp/en/rcb/baf3; Last update: 2022.07.25), and ABL proteins according to supplier’s recommendation. Briefly, total blood was collected, and 30 μl of the sample was incubated with rat anti-mouse FITC-CD45 (FITC anti-mouse CD45 Antibody, Cat# 103108, Biolegend), rat anti-mouse PE-F4/80 (PE anti-mouse F4/80 Antibody, Cat# 123110, BioLegend), and anti-ABL (c-Abl Antibody; Cat# MA5-14398; Invitrogen) antibodies for 15 min. Then, samples were incubated in red blood cell lysis solution for 30 min at 37 °C. Finally, the sample was centrifuged at 2000 rpm for 10 min. and incubated with DyLight 488 donkey anti-mouse antibody (1:500) (to identify ABL primary antibody), rinsed and resuspended for analysis on a BD LSR Fortessa II flow cytometer (BD Biosciences). Fifty thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software.

Frozen kidney sections of 4 μm thickness were fixed for 20 minutes with 4% paraformaldehyde followed by treatment with 0.2% Triton X-100 to enhance antigen permeability. After blocking in 2% rabbit serum for 60 minutes, sections were incubated overnight at 4°C with Alexa Fluor® 488 anti-mouse CD206 antibody (1 : 100, catalog no. 141709; Biolegend) and PE anti-mouse F4/80 antibody (1 : 100, catalog no. 123109; Biolegend). Ultimately, sections were washed twice with PBS and then viewed with a fluorescence microscopic Olympus IX51; 5 fields of cortical area under ×400 magnification were randomly recorded for each kidney section.

Experimental samples and single-stained controls were resuspended in 100 µL antibody master mix (FcR Block CD16/CD32 Monoclonal Antibody at 1:100, ThermoFisher 14-0161-82; Invitrogen™ eBioscience™ Fixable Viability Dye eFluor™ 780 at 1:1000; PE anti-mouse F4/80 antibody at 1:50, Biolegend #123,109; AF488 anti-mouse CD326 antibody at 1:100, Biolegend #118,210; APC/Cy7 anti-mouse CD90.2 antibody at 1:100, Biolegend #105,328) while unstained controls were incubated in FACS Buffer (2% FBS and 1 mM EDTA in PBS) for 30 min on ice protected from light. Following antibody staining, samples were fixed in 2% paraformaldehyde (PFA) for 30 min on ice. Cells were washed and resuspended in 300 µL FACS buffer. Flow cytometry was performed on a BD LSR Fortessa X-20 and data was analyzed via FlowJo.