Anti p53 do 7

For immunohistochemical analysis, 3-µm thick tissue sections were mounted on organosilane-coated slides (3-aminopropyltriethoxysilane; Sigma Chemical Co., St. Louis, MO, USA). Deparaffinization, rehydration and antigen retrieval were performed using Trilogy (Cell Marque, CA, USA) diluted in distilled water (1:100) and heated in a Pascal pressure cooker. Endogenous peroxidase and nonspecific antibody reaction were blocked with 3% hydrogen peroxide and Protein Block (Thermo Scientific, Runcorn, UK), respectively. The tissues were incubated with primary antibodies anti-CLIC4 (EPR14253, Abcam, 1:4000, 60'), anti-p53 (DO7, DAKO, 1:400, 60'), anti-TGF-β (3C11, Santa Cruz, 1:500, 60'), anti-TNF-α (52B83, Santa Cruz, 1:400, overnight) and anti-α-SMA (1A4, DAKO, 1:800, 60'). Antibodies were detected using the HiDef DetectionTM HRP Polymer system (Cell Marque) and reaction was developed with diaminobenzidine as chromogen (DAB, Sigma Chemical, St Louis, MO, USA). The sections were counterstained with Mayer's hematoxylin and mounted in Permount® (Fisher Scientific, Fair Lawn, NJ, USA). Replacement of the primary antibodies with bovine serum albumin was used as negative control and human melanoma tissue served as positive control.

Immunohistochemical analyses were performed on formalinfixed paraffin-embedded specimens. Antigen retrieval was performed by autoclaving the samples in 10 mM citrate buffer (pH 6.0) for 10 min. Anti-ARID1A (HPA005456; 1:2000; Sigma-Aldrich, St Louis, MO, USA) and anti-p53 (DO-7; 1:100; Dako, Glostrup, Denmark) antibodies were used as primary antibodies. An automated stainer (Dako) was used in accordance with the manufacturer's protocol. ChemMate EnVision (Dako) methods were used for detection. For p53 staining, diffuse and complete lack of staining were regarded as abnormal expression patterns. For ARID1A, areas devoid of expression, occupying > 10% of lesions, were regarded as significant.