Antibody diluent blocking buffer

The slides were microwaved in EDTA buffer (Opal-7 Color Manual IHC kit; PerkinElmer, Waltham, MA, USA) and cooled for 1 h. Sections were then incubated in Antibody Diluent/Blocking Buffer (Opal-7 Color Manual IHC kit; PerkinElmer) for 10 min at room temperature and then incubated with primary antibodies. Triple staining was performed with CD8/FOXP3/CD33 overnight at 4°C. Samples were washed three times in TBST for 3 min each and then incubated in Polymer HRP (Ms + Rb) (Opal-7 Color Manual IHC kit; PerkinElmer). Samples were rinsed three times in TBST for 3 min each and then incubated in Opal Fluorosphore working solution (Opal-7 Color Manual IHC kit; PerkinElmer) for 10 min at room temperature. Samples were rinsed in TBST, microwaved in EDTA buffer, and then used DAPI Counterstain (Opal-7 Color Manual IHC kit; PerkinElmer) to visualize nuclei. In triple staining, CD33 were labeled with Opal 520 Fluorosphore (Green), CD8 was labeled with Opal 570 Fluorosphore (Red), and FOXP3 was labeled with Opal 690 Fluorosphore (white). For taking photomicrographs we used ZEISS fluorescence microscope (ZEISS, Axio Imager2). The ratio of positive cells in immune cells was defined as the ratio of the number of each positive immune cells to the whole number of each immune cells (red, white, and green) in a 4-mm² field of view, from five separate areas.

Immunofluorescence analysis was performed as previously described [21 (link)]. The slides were microwaved in AR9 buffer (Opal-4 Color Manual IHC kit; PerkinElmer, Waltham, MA, USA) and cooled for 30 min. Sections were then incubated in Antibody Diluent/Blocking Buffer (Opal-4 Color Manual IHC kit; PerkinElmer) for 10 min at room temperature and then incubated with primary antibodies (listed above). Triple staining was performed with antibodies against IL-8 and SerpinE1 overnight at 4 °C and antibodies against CD206 for 2 h at room temperature. Samples were washed three times in TBST for 2 min each and then incubated in Polymer HRP (Ms + Rb) (Opal-4 Color Manual IHC kit; PerkinElmer). Samples were rinsed and then washed three times in TBST for 2 min each and then incubated in Opal Fluorophore working solution (TSA Plus System; PerkinElmer) for 10 min at room temperature. Samples were rinsed in TBST, microwaved in AR9 buffer, and then mounted with VECTASHIELD Mounting Medium For Fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA). Photomicrographs were obtained using a light microscope with a digital camera (BZ-X800 series; Keyence, Tokyo, Japan). The ratios of triple IL-8-, SerpinE1-, and CD206-positive cells in immune cells and tumor areas were quantified using the BZ-H4C and BZ-H4CM analytic applications.