Following perfusion with saline, hearts, kidneys and aortas were fixed in buffered formalin, embedded in paraffin, and sectioned according to standard procedures. All tissues were stained with hematoxylin and eosin, α-smooth muscle actin, Sirius red, F4/80, CD45, collagen 1 and collagen 3, (all Abcam, Cambridge, UK). Images were captured at 20x magnification, using a Panoramic Scanning 250 microscope and analysed blindly using Panoramic Viewer software (3D Histech Ltd., Budapest, Hungary). Five images of tissues were captured per mouse and staining was quantified as percentage of total area according to published methods using Image J software [17 (link)].
Collagen 1
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Canada
Collagen I is a naturally-occurring structural protein found in various tissues, including skin, bone, and tendon. It is a key component of the extracellular matrix and plays a crucial role in the structural integrity and function of these tissues. Collagen I is widely used in cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Abcam (72 mentions)
Collagen 3, by Abcam (49 mentions)
Fibronectin, by Abcam (38 mentions)
Tgf β1, by Abcam (25 mentions)
Gapdh, by Abcam (22 mentions)
Pvdf membrane, by Merck Group (22 mentions)
Collagen 4, by Abcam (21 mentions)
β actin, by Abcam (17 mentions)
Vimentin, by Abcam (15 mentions)
α sma, by Merck Group (13 mentions)
Ripa lysis buffer, by Beyotime (11 mentions)
P smad3, by Cell Signaling Technology (10 mentions)
E cadherin, by Abcam (10 mentions)
Tgf β, by Abcam (10 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Bca protein assay kit, by Beyotime (9 mentions)
Smad2 3, by Cell Signaling Technology (9 mentions)
Smad3, by Cell Signaling Technology (9 mentions)
E cadherin, by Cell Signaling Technology (8 mentions)
Polyvinylidene difluoride membrane, by Merck Group (8 mentions)
278 protocols using collagen 1
1
Histological Analysis of Cardiovascular Tissues
2
Aortic Valve Tissue Analysis
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After euthanasia, thoracotomy was performed to remove the heart and proximal aorta, which were frozen in optimal cutting temperature compound (10.24% polyvinyl alcohol, 4.26% polyethylene glycol, and 85.5% nonreactive ingredients). The apical two thirds of the heart was removed, and remaining tissue was secured in a cryostat. Sections, 10 μm thick, were obtained from proximal, mid, and distal aortic valve from each mouse. Slides were stained with Alizarin Red or Picrosirius Red to quantitate the amount of calcium or collagen, respectively, as described previously.12 Movat's Pentachrome stain was used to visualize proteoglycans and collagen in the aortic valve, as described previously.13 Sources of primary antibodies for immunostaining were as follows: collagen‐1 (Abcam, Cambridge, MA), α‐smooth muscle actin (α‐SMA; Abcam), Runx2 (Santa Cruz Biotechnology, Dallas, TX), and CD31 (BD Biosciences, San Jose, CA).
To detect phosphorylated EGFR in aortic valve, hearts from control mice and Velvet mice, aged 2.5 to 4 months, were incubated in DMEM in the presence (N=5 control, and N=6 Velvet) or absence (N=5 control, and N=5 Velvet) of recombinant EGF (40 ng/mL; R&D Systems, Minneapolis, MN), for 12 minutes, then frozen in optimal cutting temperature compound, cut in 10‐μm sections, and stained with anti–phosphorylated EGFR antibody (Cell Signaling, Danvers, MA).
To detect phosphorylated EGFR in aortic valve, hearts from control mice and Velvet mice, aged 2.5 to 4 months, were incubated in DMEM in the presence (N=5 control, and N=6 Velvet) or absence (N=5 control, and N=5 Velvet) of recombinant EGF (40 ng/mL; R&D Systems, Minneapolis, MN), for 12 minutes, then frozen in optimal cutting temperature compound, cut in 10‐μm sections, and stained with anti–phosphorylated EGFR antibody (Cell Signaling, Danvers, MA).
3
Epithelial-Mesenchymal Transition Pathway
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Recombinant human TGF-β1 and LiCl were purchased from Sigma-Aldrich (St Louis, Mo, USA). The DMEM-F12 medium and fetal bovine serum (FBS), were supplied by Gibco (BRL Grand Island, NY, USA). Akt1, Akt2, Akt3, p-Akt (Thr308), p-Akt (Ser473), GSK3β (glycogen synthase kinase-3β), p-GSK3β, p-Smad3 and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA). Collagen 1, α-SMA and Snail were obtained from Abcam (Cambridge, UK). TGF-β1, β-catenin, fibronectin, Vimentin and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
4
Protein Extraction and Western Blotting
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PTECs were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 mM Na3VO4] supplemented with complete protease inhibitors (Roche Applied Science, Indianapolis, IN). The kidney homogenate was centrifuged at 12,000 × g for 30 minutes at 4 °C, and the protein concentration of the supernatant was determined by the Bradford method. Different amounts of extracted protein were separated by SDS-PAGE and transferred onto Immobilon-FL 0.4-μm polyvinylidene difluoride membranes (Millipore). Tris-buffered saline containing 0.1% Tween 20 was used as the washing buffer. After probing with primary antibodies, anti-rabbit (1:5,000; Cell Signaling Technology, Danvers, MA) and anti-mouse (1:6,000 for β-actin; Cell Signaling Technology) antibodies were used as secondary antibodies. Detection of labeled proteins was performed with an enhanced chemiluminescence system (ECLTM PRN 2106; Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities were analyzed using a gel documentation system (Bio-Rad Gel Doc 1000 and Multi-Analyst version 1.1)17 (link).
Western immunoblotting was performed using primary antibodies against collagen 1 (Abcam), fibronectin (Santa Cruz Biotechnology, Dallas, TX), αSMA (Abcam), and β-actin (Cell Signaling Technology).
Western immunoblotting was performed using primary antibodies against collagen 1 (Abcam), fibronectin (Santa Cruz Biotechnology, Dallas, TX), αSMA (Abcam), and β-actin (Cell Signaling Technology).
5
Age-Dependent Penile Tissue Remodeling
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The institutional animal care and use committee at the VA San Diego Healthcare Systems approved the study protocol and all experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Ten each of wild‐type C57BL6 male young (2–3 months) and old (22–24 months) mice were subjected to the physiological studies. At the end of the study, animals were sacrificed and cavernosal tissue harvested for immunostaining and Western blot studies. The following antibodies were used: Collagen‐1 (cat# ab90395, Abcam), Active β‐Catenin (clone 8E7, cat# 05‐665 Millipore), CD 31 (PECAM‐1; cat# 553370, BD Pharmigen), Periostin (cat# SC‐398631,Santa Cruz Biotechnology), TGF‐β1 (cat# SC‐130348, Santa Cruz Biotechnology), Axin‐1 (SC‐293190, Santa Cruz Biotechnology).
6
Protein Expression Analysis in Skin
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Western blotting was performed on the original samples used for MS analysis or on new wounds (n = 3). The lysates from skin were electrophoresed, transferred to nitrocellulose, and probed for selected proteins as described earlier48 (link). The primary antibodies used in this study were TIMP1 (1: 200, cat# AF980, R&D), MMP 9 (1: 1000, cat# LS-B2486, LSBio), 14-3-3 (1:500, cat# ab155037, Abcam), actin (1:1000, cat# mAbcam 8226, Abcam), GAPDH (1:5000, cat# mAbcam 9484, Abcam), collagen 1(1:1000, cat# 34710, Abcam), collagen III (1:500, cat# sc-8781, Santa Cruz), S100A8 (1:100, cat# ab178577, Abcam), collagen XII (rabbit polyclonal, kind.pngt of Dr. D. Birk, University of South Florida and Dr M. Mark, University of Cologne, Germany). The pixel density representing each protein was determined by subtracting a background pixel density using Alphaview version 2 from FluorChem E (Proteinsimple CA, USA).
7
Renal Protein Expression Analysis
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Renal tissues or cells were harvested and centrifuged at 12,000 g to remove cell debris. Protein concentration was measured using a BCA assay kit (Pierce). Eighty micrograms of protein was subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After transfer, the membranes were blocked and blotted routinely with antibodies against VE-cadherin (BD Biosciences), α-SMA (Abcam), collagen1 (Abcam), PP2Ac (Cell Signaling Technology), occludin (Invitrogen), P-threonine/P-serine (Santa Cruz), nitrotyrosine (Cayman), acetylated-lysine (Cell Signaling Technology), phosphotyrosine (PY-20,Biolgend), PP2A,C subunit, demethylated (Millipore), vimentin (Boster) and CD31 (Proteintech). The immunolabeled proteins were detected by enhanced chemiluminescence (Pierce).The density of the bands was analyzed by Quantity One software (Bio-Rad).
8
Histopathological Assessment of Liver Tissue
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For histopathological evaluation, 10% formalin-fixed, paraffin-embedded sections of the liver were stained with hematoxylin-eosin (HE) and Sirius red. For immunohistochemical analysis, deparaffinized sections were incubated with α-SMA, collagen1 (Abcam, Cambridge, United Kingdom), PCNA and YAP/TAZ (Cell Signaling, Danvers, MA, USA). Briefly, liver tissue sections were deparaffinized, hydrated, heated to 108 °C in citrate buffer for 5 min, depletion of endogenous peroxidase activity, and then blocked with goat serum for 20 min. Next, the slides were treated with primary antibodies α-SMA (1:100), collagen1 (1:500), PCNA (1:800) or YAP/TAZ (1:100), respectively, overnight at 4 °C. An irrelevant isotype rabbit IgG was used as a negative control. The slides were incubated with secondary antibody (HRP-conjugated anti-rabbit IgG), and positive cells were visualized with diaminobenzidine (DAB). The reaction was monitored by microscopy and was terminated when properly developed.
9
Cardiac Fibroblast Protein Expression
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The proteins of heart tissue or cardiac fibroblasts were prepared in RIPA lysis (Thermo fisher Scientific, USA). The following antibodies including CREG (Abcam), αSMA (Abcam), collagen-1 (Abcam), cleaved caspase 3 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), proliferating cell nuclear antigen (PCNA, Abcam), CDC42 (Abcam), Rac1 (Abcam) and RhoA (Abcam), p21-activated kinase 1 (PAK1, Cell Signaling Technology), phosphorylated-PAK1 (Abcam) and insulin-like growth factor-2 receptor (IGF2R, Abcam) were used. β-actin was as the internal reference (Abcam).
10
Protein Expression Analysis by Western Blot
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Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer
(KeyGEN BioTECH, China) and separated by 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membranes
(Millipore, USA). After blocking nonspecific protein binding with 5% BSA, blots were
incubated with primary antibodies against CTLA4 (1:1000, R&D Systems, USA),
Collagen 1 (1:1000, Abcam, UK), Runx2 (1:1000, Cell Signaling Technology, USA) and
GAPDH (1:12000, Sanjian, China). After extensive washing with PBS containing 0.1%
Triton X-100, membranes were incubated with horseradish peroxidase (HRP)-conjugated
secondary antibody (1:5000, Zhongshan, China) for 30 min at room temperature. The
signals were visualized by ChemiDoc XRS (Bio-Rad, USA) using an Enhanced
Chemiluminescence kit (Amersham Biosciences, UK) and analyzed using the ImageJ2x
software (USA).
(KeyGEN BioTECH, China) and separated by 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membranes
(Millipore, USA). After blocking nonspecific protein binding with 5% BSA, blots were
incubated with primary antibodies against CTLA4 (1:1000, R&D Systems, USA),
Collagen 1 (1:1000, Abcam, UK), Runx2 (1:1000, Cell Signaling Technology, USA) and
GAPDH (1:12000, Sanjian, China). After extensive washing with PBS containing 0.1%
Triton X-100, membranes were incubated with horseradish peroxidase (HRP)-conjugated
secondary antibody (1:5000, Zhongshan, China) for 30 min at room temperature. The
signals were visualized by ChemiDoc XRS (Bio-Rad, USA) using an Enhanced
Chemiluminescence kit (Amersham Biosciences, UK) and analyzed using the ImageJ2x
software (USA).
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