The colon specimens were embedded in paraffin and sectioned (5 μm thickness). Slides were deparaffinized and stained with a hematoxylin and eosin (H&E) kit according to the manufacturer’s instructions (BASO, Wuhan, China). The adenoma counts of each section were examined as described by Huang et al. [35 (link),36 ]. Briefly, the count of adenomas was divided into microadenomas, low-grade macroadenomas, and high-grade macroadenomas. Sirius red staining (Abcam, Cambridge, UK) was performed according to the manufacturer’s protocol in order to evaluate the degree of colon collagen deposition and fibrosis. The degree of colon fibrosis was quantified by calculating the percentage of positive red staining area over the total colon area measured. Sirius red staining (Abcam, Cambridge, UK) was performed according to manufacturer’s protocol to evaluate the degree of colon collagen deposition and fibrosis.
Sirius red staining
Manufactured by Abcam
Sourced in United Kingdom, United States
Sirius red staining is a histochemical technique used to identify and quantify collagen fibers in tissue sections. It relies on the binding of the dye Sirius red F3B to the collagen molecules, resulting in a red-colored staining. This staining method provides a simple and effective way to visualize and analyze the collagen content in various biological samples.
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Lab products found in correlation
Masson s trichrome staining, by Merck Group (2 mentions)
Ckx41, by Olympus (2 mentions)
Universal lsabtm2 kit, by Agilent Technologies (2 mentions)
F4 80, by Cell Signaling Technology (2 mentions)
Vettest, by IDEXX (2 mentions)
Masson s trichrome staining, by Abcam (2 mentions)
α tubulin, by Merck Group (2 mentions)
T smad2 3, by Cell Signaling Technology (2 mentions)
Rabbit anti p smad2 3, by Cell Signaling Technology (2 mentions)
Rabbit anti α sma, by Abcam (2 mentions)
Oil red o, by Merck Group (1 mentions)
α sma, by Merck Group (1 mentions)
Col1a1, by Merck Group (1 mentions)
Col1a1, by Abcam (1 mentions)
Dfc295, by Leica (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Qwin 3, by Leica (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Agilent Technologies (1 mentions)
Masson s trichrome staining, by Leica (1 mentions)
9 protocols using sirius red staining
1
Histopathological Evaluation of Colon Adenomas
2
Liver Tissue Analysis for Fibrosis
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Liver tissues were fixed with 4% formaldehyde and then were dehydrated by treatment with a graded ethanol series and xylene. Sirius red staining (Abcam, Cambridge, UK) and Masson’s trichrome staining (Sigma-Aldrich, St Louis, MO, USA) of paraffin-embedded liver sections (5 µm) were used to qualitatively assess collagen deposition and the extent of fibrosis, and the procedures were carried out in accordance with the manufacturer’s instructions. The intracellular concentration of cholesterol was measured using a commercial colorimetric kit (BioVision, Mountain View, CA, USA).
3
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).
4
Histopathological Evaluation of NAFLD
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Liver tissue sections were stained with Oil Red O (catalogue no. O0625; Sigma-Aldrich) and H&E, as previously described [24 (link)]. Liver fibrosis was detected with Sirius red staining (catalogue no. ab150681; Abcam, USA) according to the manufacturer’s instructions. NAFLD Activity Score (NAS) and fibrosis stage were calculated as the sum of the scores for steatosis (0–3), lobular inflammation (0–3), hepatocyte ballooning (0–2), as assessed by H&E staining, and fibrosis (0–4), assessed by Sirius red staining [21 (link), 25 (link)]. Histological samples were anonymised and assigned a number before the slides were analysed by an experienced pathologist.
5
Investigating Hepatic Fibrosis Markers
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Cultured cells and liver tissues were lysed using lysis buffer supplemented with protease and phosphatase inhibitors. Proteins (30 µg) were separated by SDS-PAGE. Rabbit anti-p-Smad2/3 and T-Smad2/3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-α-SMA, Col1a1 and NPC2 were purchased from Abcam (Cambridge, MA, USA), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The immunoblotting signals of α-SMA and Col1a1 were normalized to that for α-tubulin (Sigma-Aldrich). The immunoblotting signals of p-smad2 was normalized to that for T-smad2 and quantified by densitometric scanning (ImageJ software, NIH, Bethesda, MD, USA). Sirius red staining (Abcam) 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. The intracellular concentration of free cholesterol was measured using a commercial colorimetric kit (BioVision, Mountain View, CA, USA).
6
Quantifying Kidney Fibrosis Using Trichrome and IHC
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Masson’s trichrome staining (Sigma-Aldrich), and Sirius red staining (Abcam, Cambridge, UK) were performed to evaluate glomerular sclerosis. For the immunohistochemical assays, paraffin-embedded kidneys were cut into 4-μm-thick slices, deparaffinized, and hydrated using xylene and ethanol. Endogenous streptavidin activity was blocked using 3% hydrogen peroxide. The deparaffinized sections were stained with an anti-KLF2 antibody for kidneys and then incubated with horseradish-peroxidase-conjugated anti-mouse IgG (DAKO, Carpinteria, CA, USA, K3954). Next, 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) was used for immunohistochemical detection. Finally, all samples were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and evaluated under a light microscope (DFC-295; Leica, Mannheim, Germany). For each sample, five fields (X400) were randomly selected, and blue- (Masson’s trichrome staining) as well as brown-stained areas (immunohistochemistry) that reflected kidney fibrosis were quantified using computer-based morphometric analysis (Qwin 3; Leica). Scoring was performed in a blinded manner using the mean values of the positive areas (%).
7
Protein and Fibrosis Analysis in Liver
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Cultured cells and liver tissues were lysed using lysis buffer supplemented with protease and phosphatase inhibitors. Proteins (30 µg) were separated by SDS-PAGE. Rabbit anti-p-Smad2/3, T-Smad2/3, p-MEK, T-MEK, p-ERK, T-ERK, p-JNK, T-JNK, p-p38, T-p38, Caspase-8, Caspase-3, Caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-α-SMA was purchased from Abcam (Cambridge, MA, USA). The immunoblotting signals were normalized to that for α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Sirius red staining (Abcam) 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.
8
Ovarian Fibrosis Detection Using Sirius Red
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Paraffin sections of ovarian tissue were exposed to xylene to remove the paraffin and rehydrated through sequential concentration changes starting with 100% ethanol, 70% ethanol, and finally, deionized water. Fibrotic areas in the ovarian tissue sections were detected using Sirius red staining (Abcam). The sections were incubated with picrosirius red solution targeting collagen for 60 min and then rinsed with acetic acid solution. The sections were dehydrated in absolute alcohol, and the slides were mounted and analyzed under a light microscope.
9
Histological and Biochemical Evaluation of Liver
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Liver tissues were fixed with 10% formalin and the sections were stained with hematoxylin and eosin (H&E) for histopathological examination. To assess the extent of fibrosis, Sirius red staining (Abcam, Cambridge, MA, USA) and Masson’s Trichrome staining (Abcam, Cambridge, MA, USA) were carried out following the manufacturer’s instructions. For immunohistochemistry staining of F4/80, liver sections were incubated with antibodies against F4/80 (1:200; Cell Signaling, Beverly, MA, USA) and detected by using the Universal LSABTM2 kit (DakoCytomation, Carpinteria, CA, USA). All sections were investigated by a light microscope (Olympus CKX41, Olympus Corp., Tokyo, Japan). Serum alanine aminotransferase (ALT), cholesterol, and low-density lipoprotein cholesterol (LDLC) were measured with a biochemical analyzer (VetTest, IDEXX, Westbrook, ME, USA).
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