Cardiac fibroblasts were divided into six groups as described in the Cardiac fibroblasts isolation and cultivation section and were then fixed with 4% paraformaldehyde for 20 min. CFs were incubated with rabbit polyclonal anti‐collagen I (1:100, Abcam) and anti‐Vimentin (1:100, CST, Beverly, MA, USA) primary antibodies overnight at 4°C and then with the DyLight 594 AffiniPure goat anti‐rabbit IgG secondary antibody (1:100, EarthOx, Millbrae, CA, USA) for 1 h at room temperature. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (1 mg/mL; Solarbio, Tongzhou District, Beijing, China) for 5 min, and images were acquired using a Leica DMi8 (Buffalo Grove, IL, USA) inverted microscope (magnification: ×200).
Anti collagen 1
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany
Anti-collagen I is a laboratory product used for the detection and quantification of collagen type I. It provides a reliable tool for researchers studying collagen-related biological processes.
Automatically generated - may contain errors
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151 protocols using anti collagen 1
1
Cardiac Fibroblast Immunofluorescence Imaging
2
Achilles Tendon Injury and Repair Dynamics
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Animals were euthanized with anesthetic overdose. Achilles tendons were isolated from surrounding tissue and dissected from both sides of hind limbs. The proximal ends of the Achilles tendons were disconnected at the end of the gastrocnemius, plantaris, and soleus, and the distal ends at the calcaneus. Left leg tendons of eight control rats and six burn rats for each time point (1, 3, 7, and 14 days) were stored in RNAlater (Qiagen), whereas right leg tendons were snap frozen for protein extraction. Samples were stored in −80°C for further analysis. Tendon RNA was obtained with RNAeasy Universal kit (Qiagen). cDNA conversion was done with iScript kit (Bio-Rad), and qPCR with SsoFast Eva Green kit (Bio-Rad). Primers for IL-6, TNF, IL-1β, col1a1, col3a1, MMP9, MMP13, and TGFβ1 were purchased from QuantiTect Primers (Qiagen). Gene expression was calculated using the ΔΔCt method with 18 s as housekeeping.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
For protein extraction, tendon tissue was homogenized in RIPA buffer (Invitrogen) with proteinase inhibitor cocktail (Sigma). Protein quantification was performed with BCA assay (Pierce); 10 μg of total protein was used for Western blot. Antibodies included anti-collagen I (Abcam), anti-collagen III (Abcam), goat anti-mouse HRP (Pierce), and goat anti-rabbit HRP (Pierce), which were prepared in 2% BSA. Blots were developed with ECL (Pierce) using a CCD camera system.
3
Arterial Protein Isolation and Analysis
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For the isolation of proteins from arterial tissues, the samples were lysed by the addition of SDS-PAGE sample buffer followed by homogenization. After transfer to PVDF membranes (Millipore, Darmstadt, Germany), proteins were incubated with primary antibodies using anti-collagen I (Abcam, ab21286, Cambridge, UK), NOTCH1, 2, and 3 (Cell Signaling, Danvers, MA, USA). Secondary antibodies specific for peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich; 1:10,000, St. Louis, MO) or anti-rabbit IgG (Sigma-Aldrich; 1:5000, St. Louis, MO) were used as needed. Blots were visualized using an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK). Samples were normalized to GAPDH (Santa Cruz; 1:10,000, SC-32233, CA, USA) and quantified by densitometry.
4
Quantitative Western Blot Analysis
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The samples of lung tissues of rats were homogenized in liquid nitrogen and the homogenate was lysed into lysis buffer on ice for 30 min (Beyotime, Jiangsu, China). For detection of protein expression in Beas-2B cells, the cells were was lysed into lysis buffer on ice for 30 min. Total protein lysates were estimated using the BCA protein assay (Beyotime, Jiangsu, China). The lysates (40 μg) of total protein per well were separated using 10% SDS polyacrylamide gel, and then transferred onto immobilon-PSQ transfer PVDF membrane (Millipore, Bedford, MA). Membranes were detected of phosphorylated forms and expression of protein. Primary antibodies were anti-phospho-Smad2, anti-TGF-β1, anti-α-SMA (1: 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Collagen I (1:3000 dilution; Abcam) and anti-Actin antibodies (1:3000 dilution; Sigma). The secondary antibody was a peroxidase-coupled anti-mouse or rabbit IgG (1:4000 dilution; Abmart). The membrane was exposed to Kodak X-OMAT film (Kodak, China), and the film was developed. Signals were quantified using Image J and normalized to controls.
5
Western Blot Analysis of Protein Markers
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HTMCs were washed twice in cold PBS. Total protein was extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS,1.0% Triton X-100, 5 mM EDTA, pH 8.0) with 10 × protease inhibitor cocktail (Roche, San Francisco, CA, USA). Total protein extracts (20-100 μg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membrane. Membranes were blocked with 5% nonfat dry milk and incubated overnight with the primary antibodies, antifibronectin, anticollagen I (Abcam, Boston, MA, USA), anti-EGFR, anti-pSTAT3, anti-STAT3, anti-pAKT, anti-AKT, anti-pERK, anti-ERK (Cell Signaling Technology, Boston, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. After being incubated with secondary antibodies, the antibody-antigen complexes were detected using the Chemiluminescent HRP Substrates (Millipore, Billerica, MA, USA).
6
Immunohistochemical Analysis of Liver Fibrosis
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Paraffin-embedded liver sections (3 μm thick) were deparaffinized in xylene and rehydrated with graded ethanol dilutions. Antigen retrieval was performed at high temperature under high pressure in sodium citrate buffer (10 mM, pH = 6.0) for 20 min. After blocking with H2O2 and 10% goat serum, the sections were incubated with anti-collagen I (1:200, Abcam, United Kingdom) or anti-α-smooth muscle actin (α-SMA, 1:200, Abcam) overnight at 4°C followed by incubation with biotin-streptavidin-horseradish peroxidase (HRP) detection system (ZSGB-BIO, Beijing, China) at room temperature. Finally, the sections were stained with a solution of 3, 3’-diaminobenzidine (DAB, ZSGB-BIO) and counterstained with hematoxylin.
7
Quantifying Liver Protein Expression
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Whole proteins from liver tissues were extracted using a protein extraction kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). 50 μg (7.2 μg/μL, 7 μL) of proteins for each sample were resolved by 10% SDS-PAGE, transferred to PVDF membrane (Merck Millipore, United States), and blocked with 5% non-fat dry milk before incubated with anti-collagen I (1:1,000, Abcam), anti-α-SMA (1:4,000, Abcam) or anti-HSC70 (1:1,000, Santa Cruz Biotechnology, United States) overnight at 4°C. Then, the blots were washed and incubated with HRP-conjugated secondary antibodies (1:20,000, ZSGB-BIO) at room temperature for 2 h. Protein bands were visualized by chemiluminescence using Western Blotting Luminol Reagent (Merck Millipore), and densitometric analyses were made using Quantity One software (v4.6.2). Protein levels were normalized against HSC70 and were shown as fold changes relative to the control group.
8
Comprehensive Protein Expression Analysis
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The primary antibodies used were anti-collagen I, anti-VEGF and anti-PDGFBB (Abcam, Cambridge, UK); anti-TSG101, anti-CD9, anti-CD63, and anti-CD81 (System Biosciences, Mountain View, CA, USA). The primary antibodies anti-CD41, anti-TGF-β, anti-bFGF, anti-cleaved-caspase-3, anti-Runx2, anti-β-catenin, anti-β-tubulin, anti-calnexin, anti-Bcl-2, anti-CHOP, anti-Bad and phosphorylated Bad (p-Bad), anti-Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2), anti-Akt and phosphorylated Akt (p-Akt) antibody were all obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PERK and phosphorylated PERK (p-PERK) antibodies were obtained from Santa Cruz Biotechnology.
9
Osteogenic Protein Expression Analysis
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The cells were washed three times with PBS three times and radio immunoprecipitation assay lysis buffer (Aspen Pharmacare Holdings Ltd.; cat. no. AS1004) was used to extract the total proteins from cells. Cell lysates (1×104) were subjected to 10% SDS-PAGE followed by determination of protein concentration by the bicinchoninic acid method. The proteins (50 µg) were then transferred onto a 10% SDS-PVDF membrane. The PVDF membrane was blocked by 5% bovine serum albumin (Abcam) at room temperature for 2 h. A chemiluminescence detection system (Canon, Inc.; cat. no. LiDE110) was then used to visualize proteins based on the provided instructions. Antibodies used were as follows: Anti-collagen I (1:500; Abcam; cat. no. ab34710), anti- alkaline phosphatase (ALP; 1:1,000; Abcam; cat. no. ab95462), anti-Osteocalcin (OCN; 1:500; Abcam; cat. no. ab93876), anti-Runt-related transcription factor 2 (Runx2; 1:500; Abcam; cat. no. ab23981) and anti-GAPDH (1:10,000; Abcam; cat. no. ab37168). All experiments were conducted in triplicate.
10
Protein Expression Analysis of MG63 Osteoblasts
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MG63 osteoblasts were harvested and rinsed with cold PBS. Cells were lysed and homogenized. Then the lyastes were spined at 14000 rpm for 10 min at 4°C. Protein concentration of total protein extracted was determined by the BCA Protein Assay (Pierce Chemicals Co., Rockford, IL, USA). The protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, MA, USA) using a semidry transfer apparatus. Membrane was probed with anti-MMP1, anti-MMP2, anti-RANKL, anti-OPG (Bioworld, GA, USA), anticollagen I, anti-OCN antibody (Abcam, MA, USA), and anti-GAPDH inner control antibody (Sigma, MO, USA). And then the blots were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The immune complexes were visualized with a chemiluminescence detection system (Amersham Bioscience, NJ, USA).
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