Anti foxp3 clone 150d

The tissues were removed and fixed in 10% phosphate-buffered formalin (Bio Optica), embedded in paraffin and sectioned at 3 μm. For histological analysis, sections were stained with periodic acid-Schiff or hematoxylin and eosin reagents. For immunofluorescence, the sections were rehydrated and, after antigen retrieval in citrate buffer (10 mM, pH 6), fixed in 2% formaldehyde for 40 min at room temperature and permeabilized in a blocking buffer containing 5% FBS, 3% BSA, and 0.5% Triton X-100 in PBS. The slides were then incubated at 4°C with primary antibodies anti-IDO1 (clone 10.1; Millipore), anti–Ki-67 (Abcam), anti-FOXP3 (clone 150D; BioLegend), anti-RORγt (clone REA278; Miltenyi), and anti-CD8 (clone 5H10-1; Cell Signaling Technology). After extensive washing with PBS, the slides were then incubated at room temperature for 60 min with secondary antibodies, anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific), and anti-Rabbit TRITC (BETHYL). Nuclei were counterstained with DAPI. Images were acquired using a microscope BX51 and analySIS image processing software (Olympus).