Sirius red (G1018, servicebio) and Masson‐Fontana staining (G1057, servicebio) were used to detect collagen deposition. Positive areas were quantified using ImageJ software. Random sections from each mouse were analysed.
Sirius red
Manufactured by Wuhan Servicebio Technology
Sourced in China
Sirius Red is a dye used in histology and cell biology for the staining of collagen fibers. It is a high-contrast stain that allows for the visualization and analysis of collagen content in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (2 mentions)
G1018, by Wuhan Servicebio Technology (1 mentions)
α sma, by Abcam (1 mentions)
Podocin, by Merck Group (1 mentions)
Sf93 20, by Thermo Fisher Scientific (1 mentions)
Masson s trichrome, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti cd31 antibody, by Wuhan Servicebio Technology (1 mentions)
Oil red o, by Wuhan Servicebio Technology (1 mentions)
Tissue tek optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Ab133566, by Abcam (1 mentions)
Ab129115, by Abcam (1 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
A 21141, by Thermo Fisher Scientific (1 mentions)
Ab78540, by Abcam (1 mentions)
Ab57713, by Abcam (1 mentions)
R37117, by Thermo Fisher Scientific (1 mentions)
A21240, by Thermo Fisher Scientific (1 mentions)
Lv805a, by Tissue Array (1 mentions)
Sc 20003, by Santa Cruz Biotechnology (1 mentions)
α sma, by Wuhan Servicebio Technology (1 mentions)
20 protocols using sirius red
1
Collagen Quantification via Histochemistry
2
Liver Histopathology and Apoptosis Analysis
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Liver samples from the left lateral lobes were fixed with 4% paraformaldehyde and embedded in paraffin. Sections stained with hematoxylin and eosin (H&E) and Sirius Red (Servicebio) were used to observe the pathological changes in liver tissues after deparaffinization and dehydration. Immunohistochemical staining was carried out by incubating the sections with cleaved-caspase 3 (C-caspase 3) (1:200, CST) and alpha-smooth muscle actin (α-SMA) (1:200, Abcam) antibody before visualization by color development with 3,3-diaminobenzidine (DAB) (Servicebio).
3
Histopathological Analysis of Kidney Tissue
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Kidney tissue specimens underwent fixation with 10% formalin (SF93-20; Fisher Scientific, Pittsburgh, PA, USA). Histological features were assessed by hematoxylin and eosin (H&E; Servicebio, Wuhan, China) and Periodic Acid Schiff (PAS; Servicebio) staining. Fibrosis was assessed by the ratio of fibrotic area to total area detected by Masson’s trichrome (Servicebio) and Sirius red (Servicebio) staining. Kidney apoptosis was examined by TUNEL (Servicebio). Kidney specimens underwent embedding in frozen optimal cutting temperature compound (Fisher HealthCare, Houston, TX, USA) and sectioning at 8 µm for immunostaining. The specimens underwent incubation with primary antibodies targeting SIRT1 (1:100; Abcam, Cambridge, MA, USA) and podocin (1:100; Sigma, Shanghai, China). This was followed by incubation with second antibodies conjugated with fluorescein isothiocyanate (1:500). For quantitation, 10 random high-power fields in mouse kidney samples were assessed with Image J (NIH, Bethesda, MD, USA).
4
Histological Analysis of Kidney Tissue
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Paraffin-embedded kidney sections were subjected to staining with H&E, SIRIUS RED, and MASSON using commercially available kits (Servicebio, Wuhan, China) for histological analysis.
5
Immunohistochemical Analysis of Tissue Samples
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Jelly pudding was purchased from Strong Food Co., Ltd. (Shenzhen, China). Rabbit anti-CD31 antibody (Catalog number: GB11063-2), rabbit anti-CD68 antibody (Catalog number: GB113109), Cy3-labeled goat anti-rabbit antibody (Catalog number: GB21303), goat anti-rabbit secondary antibody labeled with horseradish peroxidase (HRP, Catalog number: G1213), and DAPI (Catalog number: G1012) were purchased from Servicebio Biotechnology (Wuhan, China). Chloral hydrate (Catalog number: C104202) was purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Sirius red (Catalog number: GC307014) and 3,3′-diaminobenzidine (DAB) color kits (Catalog number: G1012-1) were obtained from Servicebio Biotechnology (Wuhan, China). Suture needles and nylon sutures were purchased from Golden Circle Medical Supplies Co. (Shanghai, China).
6
Comprehensive Liver Tissue Analysis
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The liver was embedded with tissue-Tek optimal cutting temperature compound (Sakura Finetek, Japan), or paraffin, and liver sections were prepared. Liver sections were used for H&E, Sirius Red, and Oil Red O staining (Servicebio, Wuhan, China). For immunohistochemistry, antigen retrieval was performed under high pressure and temperature in 0.01 mmol/L citrate buffer (pH = 6.0). Then, the sections were then incubated with 3% H2O2 at room temperature for 10 min to block the endogenous peroxidase activity. After blocked with 1% BSA, the sections were incubated with primary antibodies overnight at 4 °C and then secondary antibodies were used for 1 h at 37 °C. Immunochemical staining of collagen type I (Col1α), CD68, and 4 hydroxynonenal (4-HNE) was performed using paraffin-embedded liver sections. Detailed information about antibodies is listed in Supporting Information Table S3 . Quantitative analysis was performed using open-source software (Image-J software (http://imagej.nib.gov/ )).
7
Immunohistofluorescence of Liver Fibrosis
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Formalin fixed and paraffin embedded liver fibrosis tissue arrays were purchased from US Biomax (LV805a) (Rockville, MD). Tissue array patient information is shown in Supplementary Table 2 . IHF was performed according to a previous report34 (link). Thin cryosections (4 μm) of liver tissue were fixed in acetone for immunohistofluorescence, stained with the indicated antibodies. Antibodies used in IHF were: anti-CUGBP1 (Santa Cruz Biotechnology, SC-20003, 1: 50), α-SMA (Santa Cruz Biotechnology, SC-32251, 1: 50), Reelin (Abcam, ab78540, 1: 50), IFN-γ (Abcam, ab133566, 1: 50), Cytoglobin (Abcam, ab57713, 1: 50), CUGBP1 conjugated to Alexa Fluor 488 (Abcam, ab129115, 1: 100), IFN-γ conjugated to PE-CF594 (BD Biosciences, 562303, 1: 50), goat anti-mouse IgG2a conjugated to Alexa Fluor 594 (Invitrogen, A-21135, 1: 500), goat anti-mouse IgG1 conjugated to Alexa Fluor 647 (Invitrogen, A-21240, 1: 500), goat anti-rabbit IgG conjugated to Alexa Fluor 594 (Invitrogen, R37117, 1: 500), goat anti-mouse IgG2b conjugated to Alexa Fluor 488 (Invitrogen, A-21141, 1: 500). The sections were then stained with DAPI and examined with a confocal laser scanning microscope (Leica, Wetzlar, Germany). Sirius Red staining was performed by Servicebio (Wuhan, China). The liver fibrosis stage was assessed by Ishak scale35 (link).
8
In Vivo Compatibility of Auricle Scaffolds
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Similar to previous animal experiments, SD rats (n = 6) were divided into two groups, which were used for the in vivo compatibility study of auricle-shaped scaffolds. HDPE (auricle) and pDA-EFE (auricle) were subcutaneously implanted between the skin and fascia on the backs of the rats. After 28 days, skin biopsies, including the scaffolds, were divided into two parts for histological and immunohistochemical analyses. Tissues were fixed, dehydrated, embedded in paraffin, sectioned, and stained with H&E, CD68, Sirius red, CD31(Servicebio, China), α-SMA (Servicebio, China), and DAPI to evaluate histocompatibility and vascularization.
9
Histological Evaluation of Liver Damage
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Liver damage was evaluated by histological examination in the chick embryos, as the method described by Zhang et al. and with some modifications [13 (link)]. In brief, chicken embryo liver tissue was fixed in 4% paraformaldehyde solution for 10 days and then embedded in paraffin, then sectioned into 5 μm paraffin slices. After dewaxing and rehydrating, the slice was stained with hematoxylin and eosin (H&E) and sirius red (Servicebio, Wuhan, China). The morphology of the tissue sections was observed under an automatic scanning microscope (Pannormic, Budapest, Hungary).
10
Liver Fibrosis Tissue Histology
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Formalin fixed and paraffin embedded liver fibrosis tissue arrays were cut 5 μm. Sirius Red staining was performed by Servicebio (Wuhan, China). The liver fibrosis stage was assessed by Ishak scale.
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