Anti cd103

Xylene was used to dewax paraffin embedded colonic tissue. The tissue was rehydrated in gradient concentration of ethanol (100–70%) subsequently. Then endogenous peroxidases were removed in 3% hydrogen peroxidase for 15 min at room temperature followed by 1% goat serum albumin for 10 min at room temperature for blocking. The anti-CD103 (Abcam, Cambridge, UK) was used as primary antibody, incubated overnight at 4 °C. Then sections incubated with horseradish peroxidase-conjugated secondary antibody for 60 min at 37 °C. The sections were counterstained with hematoxylin, dehydrated with ascending concentrations of ethanol, cleared in xylene and mounted.

Deparaffinization and antigen retrieval were performed in mouse melanoma lung metastasis using a PT-link system (Dako) and stained as previously described (13 (link)) The following antibodies were used for immune stainings: anti-iNOS, anti-CD206, anti-CD103, anti-Ki67, anti-granzyme B, anti-MPO, anti-CD86, anti-CD68, anti-MHC-II, anti-CD11b, anti-Ly6C, anti-Ly6G, and anti-PD-L1 all purchased from Abcam; anti-CD11c and anti-F4/80, purchased from BioLegend; anti-Foxp3 (Cell Signaling); anti-Arg1 (Bioss) and anti-CD8 (Dako) primary antibodies, anti-CD4 (BioLegend) and anti-CD25 (R&D Systems) followed by fluorescently labeled secondary antibodies. Images were acquired using an Axio Observer Light Microscope with the Apotome.2 (Zeiss). Metastatic melanoma lesions were gated by generating a region of interest (ROI), and threshold merge fluorescence was limited to ROI and calculated using the NIS-Elements Advanced Research 4.0 software (Nikon, Tokyo).