SIS samples of 5 × 5 mm were fixed in 4% paraformaldehyde solution for 48 h at room temperature and subsequently removed to a dehydration box. The dehydration box was successively dehydrated in gradient alcohol. The scaffold was embedded in paraffin, cut into 4-µm thick sections, and stained with hematoxylin-eosin (HE), DAPI staining, Masson’s trichrome staining, and Sirius red staining, according to the manufacturer’s instructions (Servicebio, China), and were then observed under a microscope.
Sirius red staining
Manufactured by Wuhan Servicebio Technology
Sourced in China
Sirius red staining is a histochemical technique used to detect and quantify collagen fibers in tissue samples. It utilizes the anionic dye Sirius red F3BA, which binds to the basic groups of collagen molecules, allowing for visualization and analysis of collagen content.
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3 protocols using sirius red staining
1
Histological Analysis of SIS Scaffold
2
Histological Examination of Collagen Fibers
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All specimens were rinsed in descending order of density. Specimens were immersed in xylol I and xylol II for 20 min each, dipped in absolute ethyl alcohol I and ethyl alcohol II for 10 min each, then cleaned in an ethanol gradient (95% to 70%) for 5 min. Sirius red staining (Servicebio Technology, Wuhan, China) was used to examine pathological changes and the expression of collagen fibers. Tissue blocks were dehydrated in an ascending ethanol series for 5 min before being cleared in xylol and embedded in neutral balata. Light microscopy was used to examine the specimens, which were then analyzed by technicians who were blind to the groups to which the specimens belonged.
3
Histopathological Analysis of Cardiac Tissue
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To analyze the histopathological changes in the cardiac tissue, the heart was excised, and one part of the myocardium was fixed overnight in 4% paraformaldehyde, embedded in paraffin, and dehydrated in an ascending series of ethanol (70, 80, 90, and 100%). The tissue samples were embedded in paraffin wax and cut into 4–5 μm thick sections. The sections were mounted on standard glass slides and stained with hematoxylin and eosin (H&E) for 2 min before histological examination. Images of the stained sections were obtained using a light microscope (EVOSM5000, Thermo Fisher Scientific, Carlsbad, CA, USA). The degree of myocardial fibrosis was examined by Masson staining (Solarbio, Co., Ltd., Beijing, China) and Sirius red staining (Servicebio, Co., Ltd., Wuhan, Hubei, China). Moreover, dihydroethidine (DHE) staining was used to detect the production of reactive oxygen species (ROS) in the myocardium (Beyotime Biotechnology, Shanghai, China). Finally, the DHE fluorescence intensity was quantified using ImageJ 5.0 software.
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