Tsatm indirect

Paraffin sections (as illustrated in Supplementary Fig. 5a) were subjected to Masson-Trichrome-Goldner staining (Merck Millipore, Darmstadt, Germany) to label collagen fibres. For immunohistochemistry, antigens were retrieved with target retrieval solution (Dako, Hamburg, Germany) and blocked with serum derived from the same animal as the secondary antibody (Vector laboratories, Burlingame, USA). The following primary antibodies were used: CD45 (1:100; 553076, BD Pharmingen, Heidelberg, Germany), F4/80 (1:200; MCAP497, AbD Serotec, Puchheim, Germany), αSMA (1:200; ab5694, Abcam, Cambridge, UK) and CD31 (1:100; ab28364, Abcam). Appropriate secondary antibodies (BA-1100, BA-4001; Vector Laboratories) were added and amplified with TSA^TM Indirect (Perkin Elmer, Rodgau, Germany) prior to DAB-labelling (Dako). Positively stained cells or areas were quantified by two independent, blinded investigators applying 20 standardized high-power-fields (Supplementary Fig. 5b) using an Axiostar Plus Microscope (Carl Zeiss, Jena, Germany) and ImageJ software (National Institutes of Health, Bethesda, USA).

Paraffin-embedded tissues were sectioned at 5 µm thickness, dewaxed and rehydrated in xylene and graded ethanol series. For immunohistochemistry, antigens were retrieved with Target Retrieval Solution (Dako, Hamburg, Germany), blocked with serum from the same species the secondary antibody was raised in (Vector Laboratories, Burlingame, USA), and incubated overnight with the following primary antibodies: PDK4 (1:400; ab89295; Abcam, Cambridge, UK) or 8-hydroxy-2′-deoxyguanosine (8-OHdG; 1:200; NB600-1508; Novus Biologicals, Littleton, USA). The following day, appropriate secondary antibody was added and amplified with TSA^TM Indirect (Perkin Elmer, Rodgau, Germany), before DAB-labelling (Dako). Quantification of positively stained areas was carried out by two independent, blinded investigators, using an Axiostar Plus Microscope (Carl Zeiss, Jena, Germany) and ImageJ software (National Institutes of Health, Bethesda, USA).