After treatment and fixation, cardiac fibroblasts were incubated with blocking solution at room temperature. Then, diluted primary anti-α-SMA (1:100), collagen I (1:200), and collagen III (1:200) (Boster Biological Technology, Dublin, CA, USA); ki67 (1:100), PCNA (1:200) (ABclonal Technology, Wuhan, China); and the DRP1, OPA1, or RIPK3 (1:50, Cell Signaling Technology, Danvers, MA, USA) antibody were added and incubated overnight at 4 °C. After washing, the cells were incubated by IgG conjugated with Alexa Fluor 488 or Cy3 (1:500, Beyotime, Shanghai, China) without light at room temperature for 2 h followed by DAPI staining for 15 s. The cells were observed and photographed with a laser confocal microscope. The protein expression, which is considered as the fluorescence intensity, was quantified using ImageJ software.
Collagen 1
Manufactured by Boster Bio
Sourced in China, United States
Collagen I is a type of collagen protein derived from bovine sources. It is a key structural component of connective tissues, such as skin, bone, and cartilage. Collagen I provides mechanical strength and support to these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Collagen 3, by Boster Bio (4 mentions)
α sma, by Boster Bio (4 mentions)
Fibronectin, by Merck Group (3 mentions)
Ripk3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Periostin, by Proteintech (2 mentions)
Angptl4, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Cyanine3 (cy3), by Beyotime (1 mentions)
Alexa fluor 488, by Beyotime (1 mentions)
Proliferating cell nuclear antigen (pcna), by ABclonal (1 mentions)
Anti α sma, by Boster Bio (1 mentions)
P smad 2, by Boster Bio (1 mentions)
P smad 3, by Boster Bio (1 mentions)
Ecl luminescence reagent, by Biosharp (1 mentions)
Tgf β1, by Abcam (1 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Pannoramic 250, by 3DHISTECH (1 mentions)
Collagen 3, by Abcam (1 mentions)
Pannoramic midi, by 3DHISTECH (1 mentions)
17 protocols using collagen 1
1
Immunofluorescence Analysis of Cardiac Fibroblasts
2
Western Blot Analysis of Fibrosis Markers
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The protein samples (10 μL) were separated by 10% SDS polyacrylamide gel electrophoresis, and transferred to a PVDF membrane (Millipore, Billerica, USA). Next, the membranes were blocked with 5% skim milk or 5% bovine serum albumin at 25°C for 2 h and then incubated with primary antibodies in the refrigerator overnight in a shaker at 4°C. The following antibodies were used: Kir2.1 (1:1000; Abcam), α-SMA (1:1000; Boster), collagen I (1:1000; Boster), collagen III (1:1000; Boster), TGF-β1 (1:1000; Abcam), Smad 2 (1:1000; Boster), p-Smad 2 (1:1000; Boster), Smad 3 (1:1000; Boster), p-Smad 3 (1:1000; Boster), and GAPDH (1:1000; Zhong Shan-Golden Bridge, Beijing, China). Next, the membranes were incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 2 h, followed by three times wash with TBST for 10 min each. Finally, the ECL luminescence reagent (Biosharp, Hefei, China) was used to visualize the protein bands, and band density was analyzed on a Tanon-5200 gel imaging system (Tanon, Shanghai, China).
3
Comprehensive Histological Assessment of Tissue
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4 μm paraffin-embedded tissue were deparaffinized and rehydrated, stained with hematoxyline and eosin (H&E), IHC, IF, Masson's Trichrome Kit (SenBeiJia Biological Technology Co., Ltd.) and Picro-Sirius Red (SenBeiJia Biological Technology Co., Ltd.) following manufacturer protocol. For IHC, slides were incubated overnight in a humidified chamber at 4 °C with primary antibodies as followings: LKB1 (#13,031, Cell Signaling Technology; 1:250 dilution), Collagen I (#BA0325, Boster; 1:250 dilution), Collagen III (#ab7778, Abcam; 1:200 dilution), CD8 (#98,941, Cell Signaling Technology; 1:400 dilution), HIF-1α (#ab114977, Abcam; 1:250 dilution), p-FAK (Tyr397) (#44624G, Invitrogen; 1:200 dilution). The whole slides were scanned with Pannoramic 250 (3D HISTECH). The intensities of brown-colored precipitate stained with DAB were measured and quantified with IOD or cell intensity by Image Pro Plus 6 (Media Cybernetics). For IF, the FAP/α-SMA was used for activated CAF analysis (FAP: #ab53066, Abcam, 1:200 dilution; α-SMA: #ab7817, Abcam, 1:1000 dilution). CD31 (#77,699, Cell Signaling Technology; 1:100 dilution) was used for vascular analysis. The whole slides were scanned with Pannoramic MIDI (3D HISTECH).
4
Muscle Tissue Protein Expression Analysis
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The lysates of muscle tissue samples on 1 w post injury were loaded on SDS-PAGE gel and transferred to the PVDF membrane. Then CD68 (1 : 1000, Abcam), iNOS (1 : 1000, Abcam), ArgI (1 : 1000, Abcam), TNF-α (1 : 1000, Abcam), α-SMA (1 : 1000, Cell Signaling Technology), desmin (1 : 50,000, Abcam), fibromodulin (FMOD, 1 : 1000, Abcam), myogenin (1 : 1000, Abcam), MyoD (1 : 100, Santa Cruz Biotech), collagen I (1 : 1000, BOSTER), HGF (1 : 100, Santa Cruz Biotech), AKT (1 : 1000, Cell Signaling Technology), pAKT (1 : 1000, Cell Signaling Technology), and GAPDH (1 : 20,000, Santa Cruz Biotech) were probed with the membrane as the primary antibodies at 4°C overnight. The membranes were rinsed and incubated with anti-mouse or anti-rabbit second antibodies at room temperature for 50 min. The blots were visualized by exposure to chemiluminescence ECL reagents (Beyotime). The gray values of the target bands were calculated by Image J software. Each result was obtained by at least three independent experiments.
5
Extracellular Vesicle Protein Profiling
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The proteins were isolated with RIPA lysis buffer containing protease and phosphatase inhibitors, PMSF, separated by 10–15% SDS-PAGE, and transferred to a PVDF membrane (Millipore, USA). Next, the PVDF membranes were blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies against CD9 (1 : 1,000; Cell Signaling Technology), TSG101 (1 : 1,000; Boster), CD81 (1 : 800; Proteintech), calnexin (1 : 1,000; Boster), α-SMA (1 : 1,000; Boster), vimentin (1 : 1,000; Cell Signaling Technology), collagen I (1 : 1,000; Boster), periostin (1 : 1,000; Proteintech), CD31 (1 : 1,000; Arigo), Angptl4 (1 : 800; Cell Signaling Technology), cyclin D1 (1 : 800; Cell Signaling Technology), β-catenin (1 : 800; Cell Signaling Technology), and GAPDH (1 : 2,000; Cell Signaling Technology). The next day, the secondary antibodies (1 : 3,000; Cell Signaling Technology) were incubated with the membranes for 1 h at 37°C. The bands were visualized by enhanced chemiluminescent image analysis.
6
Myocardial Fibroblast Protein Analysis
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After treatment, the proteins were extracted from the myocardial fibroblast and the concentration was quantified using the BCA method. The proteins were boiled at 95 °C for 10 min and were stored in −80 °C. Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membranes. After the membranes were blocked with 5% non-fat milk for 2 h, diluted primary anti-α-SMA (1:1000), collagen I (1:500), collagen III (1:500) (Boster Biological Technology, Dublin, CA, USA); DRP1, OPA1, SIRT3, RIPK1, RIPK3, MLKL, p-MLKL (1:1000, Cell Signaling Technology, Danvers, MA, USA); and the GAPDH (1:5000, Sigma-Aldrich, St. Louis, MO, USA) antibody was added and incubated overnight at 4 °C. After washing, the secondary antibody (Beyotime, Shanghai, China) was incubated for 2 h at room temperature. The enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) solution was dropped onto the membrane to demonstrate the protein bands. The gray values of the bands were quantified using ImageJ software.
7
Quantifying Myocardial Fibrosis Markers in Rats
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The heart tissue of rats was washed with cold PBS and fixed with 10% phosphate-buffered formalin for 24 h. The myocardial fibrosis markers were detected by staining the rat heart tissue sections with antibodies against α-SMA (1:100; Boster, Beijing, China), collagen I (1:100; Boster), collagen III (1:100; Boster), and Kir2.1 (1:100; Abcam, Cambridge, UK). Six fields of view were randomly selected for microscopic observation from each specimen under a light microscope at 400× magnification. The brown sections were observed. The myocardial fibrosis markers and the percentage of Kir2.1 (brown sections) in the entire myocardial area were calculated by Image-Pro plus 6.0 image analysis software (Media Cybernetics, Silver Spring, USA). In addition, GraphPad Prism 8 software (GraphPad, La Jolla, USA) was used to count the positive immunohistochemical staining (brown staining).
8
Western Blot Analysis of Protein Expression
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Proteins were extracted from cells using RIPA buffer, separated by SDS-PAGE and electro-transferred onto the PVDF membranes. The membrane were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature, after which the proteins were probed with specific antibodies against Collagen I, cyclin D1, MMP9, FAK, phospho-FAK(Tyr397) and GAPDH (1:1,000 dilution; BOSTER, Wuhan, China). Relative expression of detected proteins was quantified and normalized to GAPDH using Image J software (National Institutes of Health, Bethesda, MD, USA).
9
Immunofluorescence analysis of cell markers
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When the density of CFs reached 70–80%, the supernatant was removed and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were blocked with 5% bovine serum albumin (BSA) for 30 min at 37°C and incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1 : 200; Boster, Birmingham, USA), vimentin (1 : 100; Cell Signaling Technology, Danvers, USA), collagen I (1 : 100; Boster), periostin (1 : 100; Proteintech, Rosemont, USA), CD31 (1 : 50; Arigo, Taiwan), and Angptl4 (1 : 100; Cell Signaling Technology) overnight at 4°C. The next day, the fluorescence-conjugated secondary antibodies (1 : 1,000; Cell Signaling Technology) were incubated with cells for 1 h at 37°C. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, USA), and the images were viewed under a fluorescence microscope (OLYMPUS DP73, Tokyo, Japan).
10
Decellularized Heart Scaffold Characterization
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After fixation with 2.5% glutaraldehyde for 24 h, the decellularized heart was dehydrated with gradient ethanol solutions and dried under vacuum condition. The ECM was then treated with spray-gold and observed with a scanning electron microscope (Hitachi S-3400N).
For the histological examination, the fresh heart and decellularized heart were fixed with 4% paraformaldehyde. 3-5 μm paraffin-embedded sections were prepared for hematoxylin and eosin (H&E) staining and immunostaining assay. For immunostaining, sections were incubated with the primary antibodies overnight at 4°C and then incubated with FITC-labeled secondary antibodies. DAPI (Solarbio, Beijing) was then used for nuclear staining. The images were recorded with a fluorescent microscope (Olympus, Japan). The primary antibodies collagen I (1 : 150, Boster, Wuhan, China), collagen III (1 : 150), fibronectin (1 : 150), laminin (1 : 150), α-actinin (1 : 200, Sigma), and cTnT (1 : 200; Sigma) were used.
For the histological examination, the fresh heart and decellularized heart were fixed with 4% paraformaldehyde. 3-5 μm paraffin-embedded sections were prepared for hematoxylin and eosin (H&E) staining and immunostaining assay. For immunostaining, sections were incubated with the primary antibodies overnight at 4°C and then incubated with FITC-labeled secondary antibodies. DAPI (Solarbio, Beijing) was then used for nuclear staining. The images were recorded with a fluorescent microscope (Olympus, Japan). The primary antibodies collagen I (1 : 150, Boster, Wuhan, China), collagen III (1 : 150), fibronectin (1 : 150), laminin (1 : 150), α-actinin (1 : 200, Sigma), and cTnT (1 : 200; Sigma) were used.
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