Sirius red

Four‐micrometer paraffin sections were stained with Masson's trichrome (Sigma‐Aldrich). Fibrosis was quantified as the relative area of blue staining (collagen) compared to the left ventricle surface, as an average of 3–4 sections per heart at the level of the papillary muscle, using ImageJ software. The anterior wall thickness of the left ventricle was measured on Masson's trichome‐stained sections as an average of 3–4 sections per heart. For Sirius Red staining of collagen, 3–4 sections per heart were incubated with 0.1% Sirius Red (Sigma‐Aldrich). Sections were photographed with identical exposure settings under ordinary polychromatic or polarized light microscopy. Total collagen content was evaluated under polychromatic light. Interstitial collagen subtypes were evaluated using polarized light illumination; under this condition, thicker type I collagen fibers appeared orange or red, whereas thinner type III collagen fibers were yellow or green. Quantifications were performed with LAS software (Leica). For quantification of myofibroblasts, sections were stained with an antibody against smooth muscle actin (αSMA, clone 1A4 Sigma‐Aldrich, dilution 1/300). Myofibroblast density was quantified using ImageJ software by examining 10 fields per section at 20× magnification, in a blinded fashion.

Tissues were harvested, fixed, embedded in paraffin, and cut into 8 µM sections. For H&E staining, hepatic slides were stained with hematoxylin solution (Solarbio, Beijing, China) for 8 min and eosin solution (WeiAo, Shanghai, China) for 40 s. For Sirius red staining, slides were stained with Sirius red (Sigma-Aldrich, USA) for 60 min and rinsed with hydrochloric acid (0.01 M) for 1 min.

Sirius Red staining was used to visualize collagen present in the adrenal capsule [12 (link),13 (link)]. Briefly, after deparaffinization and rehydration, sections were incubated with Mayer′s Hematoxylin (Merck, Darmstadt, Germany) and Celestine Blue (Merck, Darmstadt, Germany) for 8 min followed by the incubation with 0.1% (w/v) Sirius Red (Merck, Darmstadt, Germany) for 1 h, at room temperature. Sections were washed with acidified water, dehydrated, and mounted in a resinous medium.
Slides were scanned using the image acquisition software Olympus VS110 virtual slide scanning system (Olympus, Tokyo, Japan). Images were analyzed using the FIJI as performed for the ORO staining and then the area stained with Sirius Red was normalized to the perimeter of each adrenal gland, obtained by using the same software.

Tissue sections were prepared and stained with hematoxylin and eosin solution (Merck, Darmstadt, Germany) or with 0.1% solution of Fast Green and 0.1% solution of Sirius Red (Merck, Darmstadt, Germany) in 1.2% picric acid. The analysis of the specimens was conducted using the Nikon Eclipse 80i microscope (Nikon Instruments Inc., Melville, NY, USA). Histological measurements were taken in 5 randomly selected fields of each tissue section. The number of muscle fibers was calculated per mm² of muscle. To analyze muscle regeneration, normal and regenerative muscle fibers (characterized by centrally located nuclei) were counted. Fibrosis area was calculated as the percentage of the PicroSirius Red-stained collagen in the muscles per total area of tissue (Sirius Red- and Fast Green-stained tissue) using ImageJ 1.48v (NIH, Bethesda, MD, USA).

Evans Blue, triphenyl tetrazolium chloride, hematoxylin-eosin, sirius red, Triton X-100, calcium chloride, zinc chloride, and acetic acid were from Merck (Madrid, Spain). The Masson trichrome staining kit, anti-EMMPRIN, anti-CD68, HRP/DAB IHC kit, and HRP-conjugated secondary antibodies were from Abcam (Cambridge, United Kingdom). Anti-MMP-9 and anti-MMP-13 were from Santa Cruz (Heidelberg, Germany). Ketamine was from Pfizer (New York, NY); the isoflurane was from Abbvie (North Chicago, IL); the propofol was from Fresenius (Bad Homburg, Germany); the fentanyl was from Kern Pharma (Madrid, Spain); the diazepam was from Roche (Basel, Switzerland); and the amiodarone was from Sanofi Aventis (Gentilly, France).