The mouse femurs were harvested and fixed in 4% paraformaldehyde for 2 days, and decalcified uisng 10% ethylenediaminetetraacetic acid, embedded in paraffin, then cross-sectioned (5 μm) on the rotary microtome (RM2235, Leica, Germany). Hematoxylin and eosin (HE) staining was conducted as the previous study (Zhang et al. 2018 (link)). The differentiation of osteoclasts was measured by TRAP staining. Bone sections were labeled with Masson staining according to the manufacturer’s protocol (Sigma-Aldrich, Missouri, USA). Immunohistochemical (IHC) staining was performed as previously described (Zhao et al. 2021 (link)). The primary antibody against NFATc1 (sc-7294, 1:200) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Five fields were randomly selected per section and photographed.
Masson staining
Manufactured by Merck Group
Sourced in United States
Masson staining is a histological technique used to visualize collagen fibers in tissue samples. It utilizes a combination of stains, including Celestine blue, acid fuchsin, and aniline blue, to produce a blue-green coloration of collagen fibers. This staining method allows for the clear identification and assessment of the presence and distribution of collagen in various tissues.
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Lab products found in correlation
Rm2235, by Leica (1 mentions)
Sc 7294, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Abcam (1 mentions)
Sirius red staining, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
H e staining, by Merck Group (1 mentions)
Paraffin, by Beyotime (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Rotation microtome, by Thermo Fisher Scientific (1 mentions)
H e staining, by Abcam (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
7 protocols using masson staining
1
Mouse Femur Histological Analysis
2
Evaluation of Left Ventricular Remodeling and Apoptosis in Myocardial Infarction Mice
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Mice with myocardial infarction were sacrificed after 21 days of microsphere-EV treatment. Heart tissues from different groups were fixed using 4% paraformaldehyde, embedded in paraffin and sliced (thickness = 4 μm), and then subjected to Masson staining (Sigma-Aldrich, USA), Sirius red staining (Sigma-Aldrich, USA), and Alexa Fluor 488 and wheat germ agglutinin staining (Thermo Fisher, USA). Then, ImageJ software (Version 1.5.3) was used to evaluate the parameters of left ventricular remodeling (LV wall thickness, relative scar thickness, and infarct size) from Masson (Masson positive-staining area represents scar) and Sirius red staining images (Sirius red-positive areas represent fibrosis)79 (link),80 (link), and the lateral area of cardiomyocytes from Alexa Fluor 488 and wheat germ agglutinin staining images.
Furthermore, TUNEL (Sigma-Aldrich, USA) and caspase-3 (1:500, Abcam, ab184787) immunohistochemical staining were used to detect the apoptosis of cells in the heart. Quantitative analysis of apoptotic cells was calculated according to the TUNEL-positive cells and caspase-3-positive cells.
Furthermore, TUNEL (Sigma-Aldrich, USA) and caspase-3 (1:500, Abcam, ab184787) immunohistochemical staining were used to detect the apoptosis of cells in the heart. Quantitative analysis of apoptotic cells was calculated according to the TUNEL-positive cells and caspase-3-positive cells.
3
Lung Tissue Histological Analysis
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Left lung tissue fragments were excised and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri, USA) for 24 h. After the samples were dried and embedded in paraffin (Beyotime Biotechnology, Shanghai, China), the samples were sectioned into 5-μm slices. HE staining (Sigma-Aldrich, St. Louis, Missouri, USA) and Masson staining (Sigma-Aldrich, St. Louis, Missouri, USA) were performed according to the instructions. Three fields of view in each lung section were randomly selected, and the fibrosis area was quantified relative to the total lung area.
4
Liver Histopathology and Endothelial Cells in Mice Post-BMT
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On days 7, 14, and 20 after BMT, some of the mice were sacrificed. Livers were surgically removed and fixed with formaldehyde solution. Specimens were dehydrated, waxed, and sliced into 4-μm thickness by an RM2126 microtome. After H&E staining, pathologic changes were evaluated under a light microscope. Some liver slices were treated with 3% H2O2 and blocked with 1% bovine serum albumin (BSA). The slices were then incubated with primary pan-endothelial cell monoclonal antibody (MECA-32, Novus Biologicals, Littleton, MA, USA) followed by incubation with biotinylated goat anti-rat secondary antibody and ABC HRP reagent. Color was developed with 3,3′-diaminobenzidine. Quantification of MECA-32-positive stained sinusoidal ECs was performed using NIH ImageJ software and expressed as the number of positive stained cells/analyzed area. Masson staining was carried out in accordance with the manufacturer’s protocol (Sigma-Aldrich, Tokyo, Japan).
5
Decalcification, Paraffin Embedding, and Masson Staining
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The fixed femurs were decalcified by decalcifying solution with EDTA (Sigma-Aldrich, Missouri, USA). Then, the samples were embedded in paraffin. Four-micrometer sections were prepared by a rotation microtome (Thermo, Massachusetts, USA). Bone sections were labeled with Masson staining according to the manufacturer’s protocol (Sigma-Aldrich, Missouri, USA).
6
Histological Assessment of Kidney Injury
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For assessment of kidney injury, renal sections were stained with hematoxylin and eosin (H&E) and Masson. Briefly, renal tissues of each mouse were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned at 4 μm thickness. H&E staining (Abcam, UK) was performed to detect general morphological changes and Masson staining (Sigma-Aldrich, USA) was assessed to examination of matrix deposition within the interstitium according to standard protocols.
7
Cardiac Tissue Histological Analysis
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Left ventricular tissue was fixed in 4% formalin for 48 h, embedded in paraffin and cut into 5 μm slices. The left ventricular tissue slices were stained with hematoxylin/eosin (HE) staining, Masson staining, and fluorescein isothiocyanate (FITC)-labeled lectin wheat germ agglutinin (Sigma, St. Louis, MO, USA). The cross-sectional areas (CSAs) were measured in a blinded fashion with the Image Pro Plus 5.1 Image analysis program (Media Cybernetics, Silver Spring, MD, USA). The fraction of myocardial volume occupied by fibrillar collagen was calculated as the ratio of the fibrotic area to the total LV area.
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