Envision hrp kit

The paraffin-embedded sections were stepwise dewaxed from xylene, gradient ethanol to water, and performed immunohistochemical staining as protocol. IHC staining was performed with heat-induced antigen retrieval, followed by incubating in primary antibodies overnight at 4 ℃, secondary antibody (EnVision/HRP kit, DAKO) 30 min at RT (room temperature) and substrate-chromogen solution (DAB detection kit, DAKO) 5–10 min at RT, with hematoxylin counterstain for 10 s. Among each step, washing the slides in PBS three times for 5 min. Normal kidneys were used as positive controls; normal serum in place of the primary antibody as an isotype negative control, and the slides without added primary antibody were used as negative controls. The results were scored as previously described and scored as a sum of the staining intensity and percentage of positive tumor cells. Briefly, the staining intensity scaled with 0–3. Percentage of positive cells staining was scored as 0–4. The final staining was summed by intensity and percentage as 0–12. And then adapted to 4-point IRS: 0–1 (−), 2–3 (+), 4–8 (+ +), and 9–12 as (+ + +). Finally, we set “+“ as WTX expression cut off point: − as negative;+ ~ + + + as positive^{4 (link)}. The clinical pathologic meanings of the IHC data were analyzed by Chi-square analysis. IHC staining and statistics were performed in a blind manner.

IHC was performed on the TMA sections to assess the protein levels of EZH2 and NSD2 in BCs compared with benign breast lesions. Deparaffinization and rehydration, antigen retrieval, and endogenous peroxidase repression were performed on 4 μm-thick TMA slices. Then the slices were incubated with anti-EZH2 (1:500, Cell Signaling Technology, #5246) or anti-NSD2 (1:400, Abcam, #ab75359) antibody at 4°C overnight and then labeled using an EnVision HRP Kit (Dako) at room temperature for 30 min. The signal was visualized with diaminobenzidine-chromogen, followed by counterstaining with hematoxylin. Human Protein Atlas database (https://www.proteinatlas.org/) was further used to analyze the differential protein expression of EZH2 and NSD2 in BCs and normal breast tissues. The expression was defined using the criteria set in the database. Protein expression score is based on immunohistochemical data manually scored with regard to staining intensity (negative, weak, moderate, or strong) and fraction of stained cells (<25, 25–75, or >75%). Each combination of intensity and fractions is automatically converted into a protein expression level score as follows: negative—not detected; weak <25%—not detected; weak combined with either 25–75% or >75%—low; moderate <25%—low; moderate combined with either 25–75% or >75%—medium; strong <25%—medium, strong combined with either 25–75% or >75%—high.

Paraffin-embedded human tissue was deparaffinized in xylene and boiled for 20 minutes for antigen retrieval. Samples were incubated in primary pY88 paxillin antibody overnight. The sections were stained with secondary antibody for 30 min at room temperature then visualized with EnVision-HRP kit (Dako). The immunohistochemical staining was reviewed blindly by a board-certified pathologist (Dr. Willis). Stained sections were classified according to the intensity of staining and the percentage of cells showing paxillin Y88 phosphorylation staining. This was assessed in a semi-quantitative manner with assignment of staining ranging from 0 to 4+. Tumors with a score of 2+ and above are defined as high pY88 paxillin staining.

Kidney tissues from each experimental group were immersion-fixed with 4% paraformaldehyde (pH 7.4) and the sections were embedded in paraffin. Immunohistochemical staining was performed using by anti-α-SMA (1:100; Abcam, Cambridge, UK), anti collagen I (1:200; Abcam), anti Fibronectin (1:100; Abcam) and anti-E-cadherin (1:200; BD Biosciences, Bedford, MA, USA) antibodies and then detected by the EnVision-HRP kit (Dako, Carpinteria, CA, USA). All sections were counterstained with Mayer’s hematoxylin. The immunolabeling was examined under a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ, USA).

The paraffin-embedded tissues were sliced into three-micrometer sections, deparffinized, and endogenous peroxidase blocked by incubation with 0.3% hydrogen peroxide. After washing with PBS, the tissue samples were microwaved in 0.01M citrate buffer for antigen retrival, non-specific binding was blocked by treating with 5% skim milk for 30 min, and then incubated with primary antibodies against rabbit anti-RUNX2 antibody (Abcam) at 4°C overnight. After washing, the tissue samples were incubated with horseradish peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) and detected by the EnVision-HRP kit (Dako, Carprinteria, CA, USA). The immunolabeling was examined under a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ, USA).