3 3 diaminobenzidine substrate

Tissue microarray (TMA) slides were dewaxed at 55°C for 20 min followed by three 5 min washes with xylene. The tissues were then rehydrated by washing the slides for 5 min each with 100%, 95%, 80% ethanol and finally with distilled water. The slides were then heated to 95°C for 30 min in 10 mmol/L sodium citrate (pH 6.0) for antigen retrieval and then treated with 3% hydrogen peroxide for 1 hour to block the endogenous peroxidase activity. After blocking the slides with the universal blocking serum (Dako Diagnostics, Carpinteria, CA, USA), the sections were incubated overnight with monoclonal mouse anti-p300 antibody (1:50 dilution; Millipore, USA) or with mouse polyclonal anti-Braf antibody (1:100 dilution; Sigma, USA) at 4°C. The sections were then incubated for 30 min with a biotin-labeled secondary antibody and then with streptavidin-peroxidase (Dako Diagnostics). The samples were developed by treatment with 3,3′-diamino-benzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) and with hematoxylin to counter-stain the nuclei. Negative controls were done by omitting the p300/Braf antibody during the primary antibody incubation.

Tissue specimens were obtained from patched skin. The specimens were fixed in 10% buffered formalin (Duksan, Ansan, Korea) and embedded in paraffin. Multiple 4-μm sections were stained with hematoxylin and eosin and analyzed by using bright-field microscopy (Olympus, Tokyo, Japan). To analyze immune cell infiltration in the skin, sections were stained with anti-mouse CD3e polyclonal antibody (ab5690, Abcam, Cambridge, MA, USA), anti-mouse F4/80 monoclonal antibody (mAb) (#123101, BM8, BioLegend, San Diego, CA, USA), anti-mouse Gr-1 mAb (#550291, RB6-8C5, BD Biosciences, San Diego, CA, USA), and anti-mouse major basic protein mAb (MT-14.7, provided by Dr. James J. Lee; Mayo Clinic, Scottsdale, AZ, USA) overnight at 4 °C, followed by biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) or anti-rat IgG (Vector Laboratories) secondary antibody. The sections were then incubated with streptavidin-conjugated peroxidase (Vector Laboratories) and developed with 3,3′-diaminobenzidine substrate (Vector Laboratories). The immune cell infiltration was quantified^{44 (link)} using i-SOLUTION™ (IMT i‐Solution Inc, Vancouver, BC, Canada).

Tissue microarray (TMA) slides were dewaxed at 55°C for 20 min followed by three 5 min washes with xylene. The tissues were then rehydrated by washing the slides for 5 min each with 100%, 95%, 80% ethanol and finally with distilled water. The slides were heated to 95°C for 30 min in 10 mmol/L sodium citrate (pH 6.0) for antigen retrieval and then treated with 3% hydrogen peroxide for 1 hour to block endogenous peroxidase activity. After blocking the slides with the universal blocking serum (Dako Diagnostics, Carpinteria, CA, USA), the sections were incubated overnight with mouse anti-p-ATM (ser 1981) antibody (1:50 dilution; Biolegend, USA) at 4°C. The sections were incubated for 30 min with a biotin-labelled secondary antibody and then with streptavidin-peroxidase (Dako Diagnostics). The labelling was developed by treatment with 3,3’-diamino-benzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) and with hematoxylin to counter-stain the nuclei. Negative controls were done by omitting the p-ATM antibody during the primary antibody incubation.

For cryosectioned tissue, 4 µm-thick sections were fixed and incubated with primary antibodies overnight at 4°C, followed by incubation with biotinylated secondary antibodies (Vector Laboratories, USA). Sections were then incubated in Avidin:Biotinylated enzyme Complex (Vector Laboratories) for 30 min, developed with 3,3’-diaminobenzidine substrate (Vector Laboratories) and nuclei counterstained with methyl green. Slides were mounted with Fluka Eukitt® quick-hardening mounting medium (Sigma-Aldrich, USA). See Supplementary Methods for detailed procedure.

We obtained multiple 4 μm cut sections using a Leica microtome in immunohistochemical staining. The obtained sections were shifted to adhesive-coated slides. Dewaxing was performed with the TMA slides by heating at 55°C for 30 min and by three washes, of 5 min each, with xylene. Rehydration was done with the tissues by 5 min washes in 100%, 90%, and 70% ethanol and phosphate-buffered saline (PBS). Antigen was retrieved by heating the samples for 4 min 20 s in a microwave at full power in 250 ml 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase for 20 min. The sections were incubated with primary goat polyclonal anti-COX-2 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 : 100 in goat serum at room temperature for 1 h. After three washes of 2 min each with PBS, the sections were incubated with biotinylated anti-goat secondary antibody for 30 min (DAKO, Carpinteria, CA, USA). After three further washes with PBS, horseradish peroxidase streptavidin (DAKO) was added to the section for 30 min, followed by another three washes. The samples were developed for 1 min with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) and counterstained with Mayer's hematoxylin. They were dehydrated according to standard procedures and closed with coverslips.

Liver tissues of TGF-β gene-transferred mice were embedded in an optimal cutting temperature compound (Miles Inc., Elkhart, IN) and frozen in liquid nitrogen for following experiments. Five mm of cryosections were performed by using cryostats (Leica CM 1800, Nussloch, Germany). Histology and immunohistochemistry were performed as described previously [5 (link)]. For the detection of α-SMA, a mouse monoclonal antibody (Santa Cruz) was used. Signals were visualized by anti-mouse IgG, horseradish peroxidase labeled secondary antibody, and 3, 3'-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA). All sections were viewed under a microscope (Leica Mikrosysteme Vertrieb GmbH, Bensheim, Germany).