Fast red substrate system

All reagents and materials were purchased in Australia. PAR‐1 activating peptide (AP1; TFLLR‐NH₂) and PAR‐2 activating peptide (AP2; SLIGKV‐NH₂) were purchased from Auspep (Parkville, Vic., Australia). Fura‐2 acetoxymethyl ester was obtained from Thermo Fisher (Newstead, Qld, Australia). Antibodies were purchased from the following vendors: anticytokeratin (high molecular weight; 4βE12; Dako, Campbellfield, Vic., Australia), anti‐TAGLN antibody (HPA019467; Sigma‐Aldrich, Castle Hill, NSW, Australia), anti‐β‐actin antibody (ab8226; Abcam, Melbourne, Vic., Australia), anti‐αSMA (SP171; Sigma‐Aldrich), antivimentin (antiVEM, PA5‐27231; Thermo Fisher), anti‐VEGF (500‐P10‐50; Lonza, Mount Waverley, Vic., Australia), rabbit IgG isotype control and secondary antibodies (Thermo Fisher). DAPI counterstaining compound and CyQuant cell proliferation assay were purchased from Thermo Fisher. The protease inhibitor cocktail and other chemical reagents were purchased from Sigma‐Aldrich, except when specified. The Envision peroxidase system and Fast Red Substrate System were purchased from Dako. All cell culture media and reagents were purchased from Thermo Fisher, Australia, except for fetal bovine serum (FBS), which was from Sigma‐Aldrich.

Formalin-fixed tumors were dehydrated and embedded in paraffin. Subsequently, 3-μm tumor sections were prepared and placed on a microscope slide. Prior to incubation with primary antibody, paraffin sections were dewaxed in xylene and rehydrated in ethanol/water. For antigen retrieval, the tumor sections were treated at 99 °C for 25 min in citrate buffer (Target Retrieval Solution, pH 6.0, DAKO, Glostrup, Denmark). The following primary antibody was used: rabbit anti-cleaved caspase 3 (BD Biosciences). The tumor sections were incubated with the primary rabbit antibody diluted 1 : 100 in blocking buffer (PBS+20 mg/ml BSA+1 mg/ml human IgG) for 60 min at room temperature. After a PBS washing step, specific binding of the primary antibody was visualized using an anti-rabbit biotinylated secondary antibody (Southern Biotech, Birmingham, AL, USA) and streptavidin alkaline phosphatase (BioGenex, Fremont, CA, USA). The FAST-Red substrate system (DAKO) was used as the substrate for the alkaline phosphatase, which produced a red precipitate at antibody-binding sites. Sections were then counterstained with Mayer’s hematoxylin and mounted with glycerin-gelatin. A rabbit isotype control antibody was used for control staining.