3 amino 9 ethylcarbazole aec

For histological analysis, hearts were processed according to Salameh et al. (2015 (link)). Briefly, formalin-fixed hearts were embedded in paraffin and sectioned into 2 μm slices. Thereafter, heart slices were de-waxed, re-hydrated and specific primary antibodies to AIF (1:50, Santa Cruz, Heidelberg, Germany), cC3 (1:200, Cell Signaling, Frankfurt, Germany), PAR (1:600, Bio-Rad, Munich), and NT (1:50, Merck-Millipore, Darmstadt, Germany) were applied over night at 4°C. After several washing steps, secondary antibodies labeled with HRP (horseradish peroxidase) were administered for 1 h at room temperature. Thereafter, detection of signals was completed by application of the red chromogen AEC (3-amino-9-ethylcarbazole, Dako, Hamburg, Germany) according to the manufacturer's instruction. Nuclei of cardiomyocytes were counter-stained with haemalum. Specimen were embedded in glycerol-gelatin and investigated at 400x magnification with a Zeiss Axiolab microscope (Zeiss, Jena, Germany). Evaluation of cardiomyocytes was carried out by a blinded observer. We investigated right and left ventricular specimen separately and at least 50 cells per region were counted and the ratio of positive cells was evaluated in relation to the total number of counted cells. In that manner, 100 cells per heart and 600 cells per experimental group were counted.

For the detection of collagen fibers, liver specimen were fixed in 10% formalin, paraffin-embedded and stained in 0.1% Sirius-red in saturated picric acid (Chroma, Münster, Germany) using standard methods as previously described [11 (link)].
For immunohistochemical (IHC) staining of α-smooth muscle actin (αSMA), slides with sections were incubated with a mouse-anti-αSMA (clone 1A4; Sigma–Aldrich, St. Louis, USA) diluted 1:100 in Tris–buffered saline overnight. A secondary biotinylated rabbit-anti-mouse antibody, absorbed with rat serum (Dako, Glostrup, Denmark), was subsequently applied (1:300, 30 min) and complexed with streptavidin-conjugated alkaline phosphatase (1:500, 30 min; Dako). Finally, slides were developed with AEC (3-amino-9-ethylcarbazole) (15 min; Dako) and counterstained with hematoxylin. The amount of staining was evaluated by computational analysis (Histoquant; 3DHistech, Budapest, Hungary). Quantification (% of stained area) of IHC staining is expressed as mean±SEM.

For immunohistochemical staining, formalin-fixed and paraffin embedded tissue sections were first deparaffinized and rehydrated by washing with xylene (3x for 6 min each time), 100% ethanol, 96% ethanol, 70% ethanol (each 2x for 5 min each time) and finally rinsed in distilled H₂O. Antigen retrieval was performed in a pressure cooker for 15 min by covering the slides with Target Retrieval Solution (citrate buffer, pH 6.0; DAKO, Hamburg, Germany). After cooling down the slides for 30 min and washing with TBS (2x for 5 min each time), the primary antibody was applied for 1 h. Slides were washed again with TBS twice for 5 min prior to incubation with the biotinylated secondary antibody for 25 min at room temperature, followed by 2 × 5 min each time TBS washes, 10 min H₂O₂ block, 2 × 5 min each time TBS washes and Streptavidin-HRP (DAKO) incubation for 25 min. After 2 × 5 min each time TBS washes, peroxidase was detected by AEC (3-amino-9-ethylcarbazole, DAKO) followed by a haematoxylin stain.