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Masson s trichrome staining

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Masson's trichrome staining is a histological technique used to visualize different tissue components, primarily collagen fibers, in biological samples. The staining method involves the use of three dyes that selectively stain nuclei, cytoplasm, and collagen fibers, providing a clear differentiation between these structures.

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Lab products found in correlation

Universal lsabtm2 kit, by Agilent Technologies (2 mentions) F4 80, by Cell Signaling Technology (2 mentions) Sirius red staining, by Abcam (2 mentions) Ckx41, by Olympus (2 mentions) Vettest, by IDEXX (2 mentions) Picro sirius red stain kit, by Abcam (2 mentions) Coagulation tubes, by BD (1 mentions) Dfc450 c camera, by Leica (1 mentions) Ctr 6000 microscope, by Leica (1 mentions) Cellsens image acquisition software, by Olympus (1 mentions) Cm3050, by Leica (1 mentions) Oct medium, by Thermo Fisher Scientific (1 mentions) Superfrost plus glass slide, by Thermo Fisher Scientific (1 mentions) Bx51 microscope, by Olympus (1 mentions) α sma, by Agilent Technologies (1 mentions) Histological microscope, by Nikon (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Species specific fluorochrome conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)

18 protocols using masson s trichrome staining

1

Tumor Tissue Histological Analysis

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Tumor tissue was fixed in 4% neutral-buffered PFA, paraffin-embedded, and sectioned into 5-μm-thick slices for hematoxylin and eosin (H&E) staining or Masson’s Trichrome staining (Abcam) for immunohistochemical studies. The stained sections were imaged by a microscope (BX61, Neville).
Qiu N., Liu Y., Liu Q., Chen Y., Shen L., Hu M., Zhou X., Shen Y., Gao J, & Huang L. (2020). Celastrol Nanoemulsion Induces Immunogenicity and Downregulates PD-L1 to Boost Abscopal Effect in Melanoma Therapy. Biomaterials, 269, 120604.
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2

Histopathological Analysis of Mouse Liver

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The liver from each mouse was removed and fixed in freshly prepared 10% formalin. The sections were stained with hematoxylin and eosin (H&E) for histopathological examination. Sirius red staining (Abcam, Cambridge, MA, USA) of paraffin-embedded liver sections was used to qualitatively assess the collagen architecture and the extent of fibrosis in accordance with the manufacturer’s instructions. Masson’s Trichrome staining (Abcam, Cambridge, MA, USA) was carried out in accordance with the manufacturer’s instructions to investigate the collagen architecture. Paraffin-embedded liver sections were incubated with the antibodies against F4/80 (1:200; Cell Signaling, Beverly, MA, USA) and detected using the Universal LSABTM2 kit (DakoCytomation, Carpinteria, CA, USA) according to the manufacturer’s instructions. All sections were investigated by a light microscope (Olympus CKX41, Olympus Corp., Tokyo, Japan). Histopathological diagnosis of the mouse liver tissue was analyzed by gastroenterological physician. Serum alanine aminotransferase (ALT), albumin (ALB), and blood urea nitrogen (BUN) values were measured with a biochemical analyzer (VetTest™, IDEXX, USA).
Wang Y.H., Suk F.M., Liu C.L., Chen T.L., Twu Y.C., Hsu M.H, & Liao Y.J. (2020). Antifibrotic Effects of a Barbituric Acid Derivative on Liver Fibrosis by Blocking the NF-κB Signaling Pathway in Hepatic Stellate Cells. Frontiers in Pharmacology, 11, 388.
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3

Liver Histology and Cytokine Assays

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Paraffin embedded liver sections at 4 μm thickness were prepared for Masson's trichrome, immunohistochemistry (IHC), and TUNEL labeling. Masson’s trichrome staining was performed following the manufacturer’s instructions (Abcam, MA, USA) and photomicrographs were acquired using a CTR6000 microscope with a DFC450 C camera (Leica, Wetzlar, Germany). The hepatic fibrosis score was evaluated from eight randomly selected fields, as described [32 (link)]. IHC was performed as previously described [34 (link)]. A list of antibodies used in this study is included in Additional file 1: Table S3. DAB-based TUNEL assay kit (#ab206386, Abcam, MA, USA) was used to measure apoptosis in the liver according to the manufacturer’s instructions. After mice were anesthetized by 0.02 g/ml tribromoethanol (18 µl/g BW), blood was collected from the abdominal aorta using coagulation tubes (BD, NJ, USA). Supernatants were collected for the measurement of cytokines with ELISA detection kits according to the manufacturer’s instructions. A list of ELISA kits used in this study is included in Additional file 1: Table S4.
Qiu B., Zhong Z., Dou L., Xu Y., Zou Y., Weldon K., Wang J., Zhang L., Liu M., Williams K.E., Spence J.P., Bell R.L., Lai Z., Yong W, & Liang T. (2024). Knocking out Fkbp51 decreases CCl4-induced liver injury through enhancement of mitochondrial function and Parkin activity. Cell & Bioscience, 14, 1.
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4

Cardiac Tissue Preservation and Histological Analysis

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Following CO2 euthanasia, mice were perfused with 10 ml chilled PBS; hearts were dissected and placed in Optimal Cutting Temperature (OCT) medium (Thermo Fisher Scientific). Hearts were immediately flash-frozen over a dry ice and 2-methyl-butane slurry and stored at −80°C. Frozen cardiac tissue was sectioned using a cryostat (Leica CM3050 S). Coronal sections were cut at 7 μm thickness and transferred to Superfrost™ Plus glass slides (Thermo Fisher Scientific). Hematoxylin and eosin (H&E) staining was conducted according to standard methods. Masson’s trichrome staining was conducted according to the manufacturer’s recommendations (Abcam). Histologic sections were used to measure mitral valve thicknesses as described previously (15 (link)). CellSens image acquisition software (Olympus) and an Olympus BX51 microscope were used for brightfield image acquisition.
Meier L.A., Faragher J.L., Osinski V., Auger J.L., Voeller R., Marath A, & Binstadt B.A. (2022). CD47 promotes autoimmune valvular carditis by impairing macrophage efferocytosis and enhancing cytokine production. Journal of immunology (Baltimore, Md. : 1950), 208(12), 2643-2651.
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5

Immunohistochemistry Protocol for Colon, MLN, and Spleen

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For IHC staining, paraffin-embedded sections of the colons, MLNs and spleens were obtained (5-mm thickness) from each sample. After rehydration with 80% methanol, PBS and PBS with 12% bovine serum albumin (BSA), the sections were incubated with primary antibodies overnight at 4 °C. The next day, the slides were washed with TBS-Tween and incubated with secondary antibodies. Standard H&E staining and immunohistochemical staining were performed as described previously [34 (link)]. Primary antibodies were used as follows: CD4 (1: 200), CD8 (1: 300). All antibodies were purchased from Servicebio (Wuhan, China). Additionally, Masson’s trichrome staining was performed using a special kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The positively stained cells and collagen/area ratio were counted in five randomly selected fields at a magnification of 200× or 400× using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Zhang L., Ye C., Li P., Li C., Shu W., Zhao Y, & Wang X. (2022). ADSCs stimulated by VEGF-C alleviate intestinal inflammation via dual mechanisms of enhancing lymphatic drainage by a VEGF-C/VEGFR-3-dependent mechanism and inhibiting the NF-κB pathway by the secretome. Stem Cell Research & Therapy, 13, 448.
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6

Quantifying Smooth Muscle and Collagen in Penile Tissue

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To determine the ratio of SM/collagen fibril in the cavernous tissue, Masson's trichrome staining (Abcam, Cambridge, UK) was performed as described previously.19 (link)20 (link)22 (link) For each stained slide, areas of SM (stained in red) and collagen fibril (stained in blue) were analyzed. To determine SM content in the cavernous tissue, immunohistochemical staining was carried out using a primary antibody against α-SM actin (α-SMA) (1:100, Dako, Glostrup, Denmark) as described previously.10 19 (link)20 (link)
For histological studies, each stained slide was reviewed through quantitative analysis for the image of the penis comprising the corpora cavernosum half at ×40 using ImagePro Plus 4.5 software (Medica Cybernetics, Silver Spring, MD, USA). We analyzed stained slides of six rats from each group (two tissue sections per animal). An independent examiner reviewed these slides in a blinded fashion using the same standard.
Cho M.C., Lee J., Park J., Oh S., Chai J.S., Son H., Paick J.S, & Kim S.W. (2019). The effects of single versus combined therapy using LIM-kinase 2 inhibitor and type 5 phosphodiesterase inhibitor on erectile function in a rat model of cavernous nerve injury-induced erectile dysfunction. Asian Journal of Andrology, 21(5), 493-500.
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7

Lung Tissue Analysis: Fixation, Sectioning, and Staining

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At the end of the study, lungs of the mice were harvested and fixed in 100 mL of 4% paraformaldehyde for 24 h. The fixed lungs were then sliced midsagittally and embedded in paraffin. Sections (4 μm) were stained with Masson’s Trichrome staining (Abcam, Cambridge, UK) or Hematoxyline and Eosin (H&E) staining (Hematoxyline; Invitrogen, Carlsbad, CA/Eosin; Sigma-Aldrich, Louis, MO, USA) for morphological analysis and evaluation of collagen deposition.
Kim Y.S., Cha H., Kim H.J., Cho J.M, & Kim H.R. (2019). The Anti-Fibrotic Effects of CG-745, an HDAC Inhibitor, in Bleomycin and PHMG-Induced Mouse Models. Molecules, 24(15), 2792.
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8

Fibrotic Scar Quantification via Picrosirius Red Staining

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Upon completion of MRI scanning on day 21 after MI, hearts were harvested and processed for paraffin embedding, cut into Superfrost slides, and deparaffinized using standard methods. For Masson’s trichrome staining (Abcam), sections were stained according to the manufacturer’s instructions. For Picrosirius red staining, sections were stained using the Picro Sirius Red Stain Kit (Abcam) for 60 minutes according to the manufacturer’s protocol and imaged on a Nikon TE2000 microscope under transmission and polarized light. Quantification of fibrotic scarring based on (Picrosirius) yellow-orange birefringence signal was performed using Fiji software.
Vieira J.M., Norman S., Villa del Campo C., Cahill T.J., Barnette D.N., Gunadasa-Rohling M., Johnson L.A., Greaves D.R., Carr C.A., Jackson D.G, & Riley P.R. (2018). The cardiac lymphatic system stimulates resolution of inflammation following myocardial infarction. The Journal of Clinical Investigation, 128(8), 3402-3412.
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9

Tissue Sectioning and Staining Protocol

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Paraffin blocks of human tissues were cut to 5 μm thick with a microtome. Antigen retrieval was performed at 60 °C for 2 h. followed by tissue deparaffinization using xylol and ethanol serial dilution for 1 h. Then, Masson’s trichrome staining (Abcam, Berlin, Germany) was performed. Finally, the stained sections were mounted with vitrocloud to allow the coverslip to attach to the sample. Overviews and 20x images of stained tissues were acquired using Vectra 3.0 Microscope Automated Quantitative Pathology Imaging. Frozen mouse tumor tissue was cryosectioned as 8 μm thick slices and stained for different markers using specific primary and secondary antibodies. The immunohistochemistry protocol included a pre-washing step with DPBS for 2 min and fixing with 80% methanol for 5 min and with acetone for 2 min. The primary antibodies were applied, and samples were incubated for 1h at room temperature. This was followed by triplicate washing to eliminate unbound antibodies. Then, secondary antibodies were applied for 30 min, at room temperature. Washing steps with DPBS were again applied after secondary antibody staining. Finally, slices were covered by a coverslip using Mowiol for embedding and stored at 4 °C for further fluorescence microscopy analysis.
Tezcan O., Elshafei A.S., Benderski K., Rama E., Wagner M., Moeckel D., Pola R., Pechar M., Etrych T., von Stillfried S., Kiessling F., Weiskirchen R., Meurer S, & Lammers T. (2023). Effect of Cellular and Microenvironmental Multidrug Resistance on Tumor-Targeted Drug Delivery in Triple-Negative Breast Cancer. Journal of controlled release : official journal of the Controlled Release Society, 354, 784-793.
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10

Histomorphological Evaluation of Implant Site

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Following euthanasia, the implant sites with surrounding tissue were removed and immersed in 10% buffered formalin phosphate solution for 48 h. 10-µm-thick sections were deparaffinized twice in fresh xylene for 8–10 min and rehydrated sequentially with decreasing ethanol concentrations (100%, 95%, 90%, 80%, 70%) and distilled water (8–10 min for each step) and then stained by Masson’s Trichrome Staining (Abcam) and finally analyzed by Nikon Histological Microscope. For the immunohistochemical analysis, slides were pretreated using a standard cycle of pressure cooker to unmask epitopes in antigen retrieval solution (0.1 M sodium citrate, pH 7.2). The slides were blocked for 1 hr at RT with 10% normal goat serum (NGS) and 0.03% Triton and then incubated at 4°C overnight with anti-macrophage antibody (ab7429 abcam). Slides were then washed three times in PBS and finally mounted with ProLong® Gold Antifade Reagent (with 4′,6-diamidino-2-phenylindole (DAPI)) (Invitrogen). Slides were stored at 4°C in the dark until imaging was performed by a Nikon Histological Microscope and the fluorescence quantification by Nikon Element software.
Taraballi F., Corradetti B., Minardi S., Powel S., Cabrera F., Van Eps J.L., Weiner B.K, & Tasciotti E. (2016). Biomimetic collagenous scaffold to tune inflammation by targeting macrophages. Journal of Tissue Engineering, 7, 2041731415624667.
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