After myocardial infarction and four weeks of reperfusion, hearts were either paraffin embedded or the raw material was snap frozen at -40°C in isopentane. In paraffin sections (4 µm) the wax was dissolved by an organic solvent and the tissue slices rehydrated before picrosirius red stain was applied. Cryosections (8 µm) from raw snap frozen tissue were fixed in 4% paraformaldehyde (PFA) in 0.1M sodium phosphate buffer (PB) pH 7.4 or Zambonis fixative (0.1 M PB, 4% (w/v) PFA, 15% (v/v) picric acid) for 10 min. Washing steps were performed in PBS, PBS/0.1% Saponin or in PBS/0.2% Tween 20 according to the further requirements. Picrosirius red (SR) staining was performed according to the protocols of Junqueira et al.3 (link) and Sweat et al.5 (link)Wheat germ agglutinin (WGA) labeling was routinely used in combination with the secondary antibody in immunohistochemical preparations. Lectin from triticum vulgaris FITC conjugate (# L4895, Sigma-Aldrich, St. Louis, MO, USA) was diluted 1:100 (10 µg/mL) in the required buffer. Incubation time was one hour protected from light. After three washing steps, the sections were coverslipped with a water-soluble antifading mounting medium. Collagen I staining was performed using an anti-collagen I antibody (ab34710, Abcam, Cambridge, UK) diluted 1:100 in the required buffer. As secondary antibody anti-Rabbit-Cy3 (111-165-144, Jackson Dianova, Hamburg, Germany) was used in a concentration of 2.5 µg/mL.
Slides were analyzed with fluorescence microscope Keyence BZ 9000 (Keyence, NeuIsenburg, Germany). All shown images were taken with 4x objective using the merge function, as not otherwise specified.
Slides were analyzed with fluorescence microscope Keyence BZ 9000 (Keyence, NeuIsenburg, Germany). All shown images were taken with 4x objective using the merge function, as not otherwise specified.
