Rabbit anti mouse inos

The expressions of the M1 marker iNOS and the M2 marker CD163 were evaluated by immunofluorescent staining assays to evaluate the polarization of macrophages. iNOS and CD163 were labeled with rabbit anti-mouse iNOS (1:250, Abcam, USA), mouse anti-mouse CD163 (1:250, Bioss, China), and donkey anti-rabbit Alexa Fluor 555 (1:250, Bioss, China) and simultaneously stained with mouse anti-mouse CD163 (1:250, Bioss, China). The analysis of immunofluorescence intensity was performed as previously described.
The relative mRNA expression of anti-inflammatory cytokines was also detected by PCR with the abovementioned method. The sequences of the primers of IL-18, CD206, IL-10 and iNOS are provided in Table S1.
TNF-α, NF-kB, and IL-10, which are inflammatory-associated cytokines, were detected by enzyme-linked immunosorbent assay kits (Cusabio, Wuhan, China). After culture for 3 days as previously described, the supernatants of macrophages were collected after centrifugation at 1000 g for 10 min. Concentrations of TNF-α, NF-kB, IL-10 in the supernatant were quantified using an enzyme-labeled instrument (Epoch; BioTek Instruments, Inc., Winooski, VT, USA) according to the manufacturer’s instructions.

Antibodies against the following extracellular markers were used for flow cytometry: CD11c, CD11b, MHCII, F4/80, CD3, CD4, CD19, CD103, and Ly6C (all BD Bioscience, Erembodegem, Belgium). Footpad, pLN, and spleen cells (1 × 10⁶) were stained with antibody cocktails in PBS containing 1% BSA/1% rat serum for 20 min at 4°C followed by fixing in 2% [w/v] PFA. For intracellular cytokine staining of dendritic cells, popliteal lymph node cells from L. major infected mice were seeded at 2 × 10⁶ cells/well, surface-stained, fixed and permeabilized, and stained intracellularly for iNOS using rabbit anti-mouse iNOS (Abcam) with goat anti-rabbit PE (Abcam) and goat anti-mouse arginase (Santa Cruz Biotechnology) with donkey anti-goat PE (Abcam). Staining specificity was verified by isotype-matched antibody controls and compensation performed with BD compensation beads. Acquisition was performed using BD LSRFortessa (BD Biosciences), and data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).