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Tumor and/or normal tissue specimens from the patients were obtained at TURBT and/or cystectomy. Tissue microarrays were constructed by harvesting 2 mm tissue cores (or 1mm tissue cores when only small samples were available) from FFPE tumor or normal tissue samples of BCG-resistant patients. For IHC, the microarray sections (4-μm-thick) were mounted on glass slides, heat-treated for 15 min, and then incubated with rabbit mAb against PD-L1 (clone E1L3N; Cell Signaling Technology, Danvers, MA), mouse mAb against PD-L2 (clone MIH18; eBioscience, San Diego, CA), rabbit polyclonal Ab against CD8 (Abcam, Tokyo, Japan), rabbit mAb against FOXP3 (clone SP97; Abcam), or rabbit polyclonal Ab against CD69 (Abcam) for 30 min, followed by their corresponding secondary antibodies for 30 minutes, with the use of HISTOSTAINER (Nichirei Biosciences Inc., Tokyo, Japan). This automated system used 3, 3′ diaminobenzidine (DAB) as the chromogen (Nichirei Biosciences Inc.). FFPE tissue samples from recurrence-free patients were also sectioned and stained with anti-PD-L1 antibody (E1L3N). PD-L1- or PD-L2-overexpressing HEK293 cells (ATCC, Manassas, VA) that were transiently transfected with PD-L1 or PD-L2 cDNA were prepared as a positive control for staining of each antibody (Supplementary Figure 1).

The tissues were fixed with 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin. Sections were taken from the central portion of the wound and stained with hematoxylin-eosin (HE) according to the standard method.
For immunohistochemical analysis, after endogenous peroxidase was blocked with methanol/hydrogen peroxide, the sections were incubated with 10% normal rabbit serum for 20 min to block non-specific binding and then stained with anti-α-smooth muscle actin (α-SMA) antibody (dilution 1:200; Vector Laboratories, Inc., Burlingame, CA, USA), anti-Ly6G Ab (clone 1A8; dilution 1:100; BioLegend), or anti-MMP-2 (dilution 1:200; Chemicon, Darmstadt, Germany). The sections were incubated with peroxidase-conjugated secondary Ab (4 µg/mL; Histofine Simple Stain MAX-PO, Nichirei Bioscience, Tokyo, Japan), then reacted with 3, 3-diaminobenzidine (DAB) (Nichirei Bioscience) or Alkaline Phosphatase (Dako, Bettingen, Switzerland). The number of myofibroblasts and neutrophils in six random fields (each 0.2 mm²) was determined by counting the number of α-SMA-positive cells or the number of Ly6G-positive cells, respectively. All analyses were performed under blinded conditions.

Antibodies against CD3 (clone F7.2.38; DAKO, Glostrup, Denmark) for T cells and CD68 (clone ED1; Bio-Rad, Hercules, CA, USA) for macrophages were used for immunohistochemistry. Deparaffinized sections were pretreated with microwave in Tris-EDTA buffer (pH 9.0) and with proteinase K (DAKO) (100 μg/mL, 37 °C, 10 min) for CD3 and CD68, respectively. Sections were then incubated with 5% skim milk in phosphate buffered saline (PBS) for 15 min and with primary antibody for 1 h, followed by 1 h incubation with peroxidase-conjugated secondary antibody (Histofine simple stain MAX PO; Nichirei Biosciences, Tokyo, Japan). Positive reactions were visualized with 3,3′-diaminobenzidine (DAB; Nichirei Biosciences; Tokyo, Japan). Sections were lightly counterstained with hematoxylin. In order to evaluate hepatocellular apoptosis, liver sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method as previously described [19 (link),20 (link)]. The number of TUNEL-positive apoptotic hepatocytes was counted; more than 500 hepatocytes were analyzed in each animal to obtain reliably quantitative data.