Masson s trichrome

Referring to prior research [32 (link)], following fixation with 4% paraformaldehyde and paraffin-embedding, the cardiac tissues were sliced into sections (thickness of 5 μm) and cultured with hematoxylin and eosin solution (H&E, Sigma, St Louis, MO, USA) and Masson’s trichrome (Polysciences, Bay Shore, N.Y., USA) for observation of the myocardial histopathological structure and myocardial fibrosis.
All cardiac sections were collected and stored digitally using a Nikon slide scanner. To evaluate myocardial fibrosis, 20X Massons-Trichrome images of the entire LV were exported, and the Image-J software (National Institutes of Health, Bethesda, MD, USA) was adopted to calculate the collagen fraction (%) as the ratio of the total fibrosis area (blue) to the total tissue area. The above-mentioned experiments were performed by independent technicians who were blinded to mice grouping.

Primary cultured hDPCs were differentiated for 21 days in vitro. Differentiated cells (2 × 10⁶) were mixed with 100 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Biomet, 00110900612) alone or with either rCPNE7 (1 μg), 3-MA (40 mM), rCPNE7 + 3-MA, or rapamycin in a 0.5% fibrin gel. The cells were then transplanted subcutaneously into immunocompromised mice (NIH-bg- nu-xid; Harlan Laboratories) for 6 weeks. For the histomorphometric analysis of the newly formed mineralized tissue, samples were harvested and fixed in 4% PFA, decalcified in 10% EDTA (pH 7.4; Georgia Chem, ED2041), embedded in paraffin, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome (Polysciences Inc, 25088).

A portion of the cardiac muscles were embedded in Tissue-Tek O.C.T. Compound (Sakura) and multiple thin sections (10 μm) were cut using a cryostat (Microm). Subsequently, the sections were stained with hematoxylin and eosin (H&E), Masson's Trichrome (Polysciences, Inc.) or using immunofluorescence techniques. Masson's Trichrome staining was carried out according to the manufacturer protocol with the addition of an one hour 10% formalin fix at room temperature (RT) prior to the fixation in Bouin's solution [50 ]. For immunostaining, the sections were fixed with 4% paraformaldehyde for 15 minutes at RT then blocked with 5% BSA/0.3% TritonX-100/PBS for one hour at RT, incubated with the primary antibody, 8-oxoG DNA Lesion (Santa Cruz), overnight at 4°C and incubated with secondary AlexFluor antibody (Invitrogen) at RT for 1 hour. Slides were mounted with Vectashield Hard Set with Dapi (Vector Laboratories). All images were taken with an Eclipse Ni microscope (Nikon).

Hearts were excised, rinsed in PBS, quickly blotted on gauze, and then fixed in 10% formalin for 24 h. The biventricular apex of the heart was embedded in paraffin and cut in 4 µm sections. Sections were stained with Masson’s trichrome (Polysciences, Inc., Warrington, PA, USA) to assess collagen abundance. Stained sections were scanned (20 × magnification) with AxioScan Z1 (Carl Zeiss, Jena, Germany), to obtain whole cross-sections for collagen quantification. Total fibrosis area (%) and perivascular fibrosis (ratio of the area of fibrosis surrounding the vessel wall to the lumen area) were quantified using ZEN2 blue edition (Carl Zeiss). All histological quantifications were independently performed by trained researchers blinded to groups.

After dissection, the muscles were weighed, and gross measurements were taken. The muscles were then divided into segments, coated in tissue freezing medium, and frozen in dry ice-chilled isopentane. Frozen samples were cryosectioned at 10μm and then stained with hematoxylin and eosin (H&E) and Masson’s trichrome (Polysciences Inc., cat. no. 25088–1) to examine morphological characteristics of the SMUs. Cross-sectional samples were also immunohistochemically stained to identify myosin heavy chain (mouse monoclonal, DSHB, cat. no. MF-20c, 1:200 dilution), laminin (rabbit polyclonal, Abcam, cat. no. ab7463, 1:200 dilution), perilipin (rabbit polyclonal, Abcam cat. no. ab3526, 1:200 dilution), fast myosin isoform (rabbit polyclonal, Abcam cat. no. ab91506, 1:200 dilution), and slow myosin isoform (mouse monoclonal, Abcam cat. no. ab11083, 1:200 dilution) using a protocol described previously [23 (link),33 (link)–35 (link)]. Longitudinal samples comprising the entire width of the ZM and ~2cm of the length were sectioned at 25μm and immunohistochemically stained for acetylcholine receptors (α-bungarotoxin, 1:2000 dilution, Life Technologies, cat. no. B1601), synaptic vesicle protein-2 (mouse monoclonal, DSHB, cat. no. SV2c, 1:300 dilution), and neurofilament (mouse monoclonal, BioLegend, cat. no. 837904, 1:1000 dilution) to identify the presence of neuromuscular junctions.