Fitc conjugated anti cd206 antibody

Lung tissue specimens obtained from each tumour-resected mouse were fixed with 4% formalin and embedded in paraffin. For histological comparison, 6-μm-thick tissue sections were made and stained with hematoxylin and eosin (H&E). For immunofluorescence staining, fixed tissue sections were initially immersed in boiling sodium citrate buffer (0.01 M sodium citrate buffer, pH 6.0) for 30 min. Lung tissue sections were blocked with 5% nonfat milk, and incubated with anti-FSP-1 antibody (1:200 dilution; Millipore; catalogue number: 07-2274), FITC-conjugated anti-CD206 antibody (1:200 dilution; Biolegend; catalogue number: 141703) or PE-conjugated anti-IL-25 antibody (1:100; R&D; catalogue number: IC12991P) in 1% nonfat milk for 1 h at room temperature. Sections were then washed with PBS containing 0.1% Tween 20. To detect primary antibodies, some sections were incubated with FITC–conjugated anti-rabbit-IgG (1:200; Jackson Immunoresearch, West Grove, PA; catalogue number: 111-097-103) for FSP-1. 4′,6-Diamidino-2-phenylindole dihydrochloride (1 μg ml⁻¹; Sigma-Aldrich) was used to stain the nuclei. Fluorescence microscopy evaluation of immunostained tissue sections was performed using a Zeiss Axiovert 200 M microscope (Carl Zeiss, Heidelberg, Germany). Images were captured with a digital camera (Orca ER, Hamamatsu) and processed using Axiovision 4.6.3 (Carl Zeiss).

1×10⁶ cells/ml immature BMDCs were stimulated with CsTPs (20 μg/ml or 40 μg/ml) or 0.5 μg/ml Alb in the presence of 1 μg/ml LPS for 24 h. Receptors expressed on BMDCs including, toll like receptors (TLR) TLR2 and TLR4, C-type lectin receptors mannose receptor (MR), DC-SIGN and Dectin-2 were analyzed by RT-PCR. The primer sequences were listed in Table 1. MR was also assessed by FACS using FITC-conjugated anti-CD206 antibody (BioLegend, Canada). To block MR, BMDCs were incubated with 0.1 mg/ml or 1 mg/ml mannan (Absin Bioscience Inc, China) in complete RPMI-1640 medium for 30 min at 37°C prior to addition of the above indicated reagents.

Flow cytometry was carried out to analyze the abundance of the M1 marker CCR7, M2 marker CD206, and general macrophage marker F4/80. After 24 h of culture, BMDMs in the four groups were scraped, washed, blocked for 15 min with Blocking Buffer (Beyotime), and then stained for 1 h with an allophycocyanin (APC)-conjugated anti-CCR7 antibody (1:100, BioLegend, USA) or FITC-conjugated anti-CD206 antibody (1:100, BioLegend, USA). A PE-conjugated anti-F4/80 antibody (1:100, BioLegend, USA) was used to label all macrophages. The cells were analyzed using a BD flow cytometer with FlowJo software.