Fitc anti mouse cd86 antibody

For intracellular staining, the cells were blocked by blocking buffer (PBS solution with 5% BSA) and incubated with FITC anti-mouse CD86 antibody (Biolegend, 105005, 1:100) and APC anti-mouse CD206 (MMR) antibody (Biolegend, 141707, 1:100) at 4 °C for 1 h in a dark place. The cells were then washed in PBS and detected by a flow cytometry (BD FACSCelesta, USA). The results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

The tissue samples from the animal model were harvested and frozen immediately. The frozen tissues were sectioned by the cryostat (Leica CM1850) with 10 μm thickness and sections were attached to the poly-L-lysine-coated slides. After the slides were washed with PBS, the samples were treated with FITC anti-mouse CD86 antibody (1:200, Biolegend, 105006), CD68 antibody (1:200, Biolegend, 130712), CD19 (1:50, eBioscience, 53-0194-82), CD11c (1:50, 43-044 for 30 min, GLUT1 (1:200, Invitrogen, MA5-31960) conjugated with Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (1:500, Invitrogen, A32732) and Hoechst33342 (1 μg/mL) for 5 min prior to the experiment. The fluorescence images were obtained by Axio Observer (ZEISS, Oberkochen, Germany). The data were analyzed by the ImageJ (1.53) software.

Flow cytometry was performed as previously described (Zhao et al., 2018 (link)). Briefly, kidneys were weighed and minced. A collagenase solution (1 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) was used for renal digestion for 30 min at 37°C. A 100-μm cell strainer (Fisher Scientific) was used, together with a 1-ml syringe plunger, to acquire single cells. Cell pellets were washed and resuspended for further experiments. Anti-Mouse CD16/CD32 (553141; RRID:AB_394656, clone 2.4G2; BD Biosciences, San Jose, CA, United States) was used for non-specific Fc block. Antibodies used in this assay were acquired from BioLegend, San Diego, CA, United States; they include BV421 anti-mouse CD45 (RRID:AB_2562559), APC anti-mouse F4/80 antibody (RRID:AB_893481), PE anti-mouse CD206 (MMR) antibody (RRID:AB_10895754), FITC anti-mouse CD86 antibody (RRID:AB_313149), PerCP/Cyanine5.5 anti-mouse/human CD11b (RRID:AB_893232), APC/Cyanine7 anti-mouse CD3 antibody (RRID:AB_2242784), FITC anti-mouse CD4 antibody (RRID:AB_312713), and APC anti-mouse CD8a antibody (RRID:AB_312750). Data were acquired on a FACS Calibur cytometer [Becton Dickinson (BD), Bedford, MA, United States] and analyzed using FlowJo software (Tree Star, Ashland, OR, United States).