Fibroblasts were harvested from 6-well plates and washed thrice with PBS. The cells were fixed in 4% paraformaldehyde for 10 min at RT and permeabilized with 0.2% Triton X-10. Cells were then blocked with 5% bovine serum albumin (BSA; Amresco, LLC, Solon, OH, USA) at RT for 30 min, followed by incubation with anti-type I collagen antibody (Abcam) at 4°C overnight. Following washing thrice with PBS, the cells were incubated with FITC-conjugated secondary antibody (1:400; cat. no. ab97022; Abcam) for 30 min in the dark. Representative images were captured under a fluorescence microscope (magnification, ×400; Nikon 80i; Nikon Corporation, Tokyo, Japan).
Anti collagen type 1 antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-collagen type I antibody is a laboratory reagent used for the detection and analysis of collagen type I in various biological samples. It is a specific antibody that binds to the collagen type I protein, allowing for its identification and quantification through various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (2 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
Anti nf κb, by Abcam (1 mentions)
Anti itgb1 antibody, by Abcam (1 mentions)
Hrp labeled goat anti rabbit secondary antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Hematoxylin, by Solarbio (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Bisbenzimide, by Merck Group (1 mentions)
Anti lox antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by ZSGB-BIO (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti clusterin, by Santa Cruz Biotechnology (1 mentions)
Anti pai 1, by BD (1 mentions)
Recombinant human ang 2, by Merck Group (1 mentions)
Anti phospho smad3, by Santa Cruz Biotechnology (1 mentions)
Anti phospho smad3, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
12 protocols using anti collagen type 1 antibody
1
Immunofluorescent Staining of Collagen I
2
Immunohistochemical Analysis of Collagen, Integrin, and NF-κB
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Paraffin sections were dewaxed, and treated with sodium citrate antigen retrieval. Then tissue sections were blocked with 5% goat serum in PBST for 30 min at room temperature, and incubated with primary antibodies: anti-type I collagen antibody (1:200, Abcam, UK), anti-ITGB1 antibody (1:300, Abcam, UK) and anti-NF-κB (1:500, Abcam, UK) for overnight at 4 °C. For immunohistochemistry, samples were then incubated with goat anti-rabbit secondary antibodies (Abcam, UK) and stained with DAPI (Solarbio, China). For immunofluorescence, samples were then incubated with HRP labeled goat anti-rabbit secondary antibodies (Abcam, UK) and stained with hematoxylin (Solarbio, China). The intensity of protein expression was analyzed by image pro plus 6.0 software (USA).
3
Collagen Electrophoresis and Immunoblotting
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Electrophoresis was carried out as described by Laemmli [40 ], using 8% separating gels, which were 1.5mm thick. Samples containing 20.7μg purified collagen and human commercial collagen (Millipore, MA, USA) as control were boiled at 100°C for 5 minutes prior to gel loading. Gels were electrophoresed at 100V (constant voltage) followed by transfer onto a polyvinylidene fluoridemembrane (PVDF, 120V, 40 minutes). After transfer, PDVF membranes were immunoblotted with anti-type I collagen antibody (1:200), and 10% normal goat serum (Abcam, Cambridge, UK) as a blocking agent, overnight at 4°C. Proteins were visualized using a Fusion FX7 chemiluminiscence detection system (VilberLourmat, Torcy, France).
4
Quantitative Analysis of Type I Collagen
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Skin biopsy specimens were pulverized using nitrogen, harvested and homogenized in lysis buffer (50 mM, Tris-HCL pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors. The Western blotting was carried out as described previously.13 (link) The primary polyclonal antibody used was anti-type I collagen antibody (1:1000, Abcam, La Jolla, CA, USA). GAPDH primary polyclonal antibody was used as an endogenous control (1:1000; Santa Cruz Biotechnology). A goat anti-rabbit secondary antibody was used for 1 h (1:5000; Sigma). The reaction was revealed with chemiluminescent detection reagents (Pierce) and visualized using digital imaging equipment (ImageQuant LAS 4000, GE Healthcare). Optical density (OD) measurements were performed with NIH ImageJ 1.37 (National Institutes of Health, Bethesda, MD) to analyze the scanned membranes. Data are presented as total protein expression normalized to GAPDH. For accuracy, the experiments were performed twice, in the same conditions, for each sample.
5
Immunofluorescence Analysis of Chondrocytes
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The nasal primary chondrocytes treated with BC (1% w/v) and CS (1% w/v) for 10 days and maintained for two and four passages in culture (p2 and p4), were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X‐100 in PBS. The cells were then incubated with anti‐type I collagen antibody (Abcam) overnight at 4°C followed by a corresponding secondary antibody for 2 h at room temperature. Nuclei were stained with 2′‐(4‐hydroxyphenyl)‐5‐(4‐methyl‐1‐piperazinyl)‐2,5′‐bi‐1H‐benzimidazole trihydrochloride hydrate, bisBenzimide (Hoechst, Sigma–Aldrich). Fluorescence images were captured using the confocal system (Zeiss).
6
Immunohistochemistry of Colorectal Cancer
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For immunohistochemical staining, 4-μm sections of the human colorectal cancer tissue arrays were used. Briefly, the slides were subsequently dewaxed, rehydrated, and incubated in 3% peroxide-methanol at 37 °C for 30 minutes to quench endogenous peroxidase. Next, the sections were treated with 10% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) to block the non-specific binding and incubated with anti-collagen type I antibody (1:100 dilution, Abcam, Cambridge, CB, UK) or anti-LOX antibody (1:100 dilution, Abcam) overnight at 4 °C. The slides were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (ZSGB-BIO, Beijing, China) at 37 °C for 50 minutes. All the sections were stained with diaminobenzidine solution (DAB, Dako Cytomation, Hamburg, Germany) and counterstained with hematoxylin. The images were analyzed to quantify the LOX expression using IPP (version 6.0) under a 400× objective field. The images were evaluated by two experimenters.
7
Recombinant Ang II and Antibody Assay
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The recombinant human Ang II was purchased from Sigma (St. Louis, MO). The anti-plasminogen activator inhibitor-1 (PAI-1) and anti-fibronectin antibodies were purchased from BD Biosciences (San Jose, CA). The anti-collagen type I and anti-GFP antibodies were purchased from Abcam (Cambridge, UK). The anti-actin antibody was purchased from Sigma. The anti-clusterin and anti-phospho-Smad3 antibodies for immunohistochemical staining were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–PAI-1 and anti-fibronectin antibodies were purchased from BD Biosciences (San Jose, CA). Anti–collagen type I antibody was purchased from Abcam (Cambridge, UK). Anti-phospho-NF-κB p65, anti-phospho-NF-IκBα, anti-IκBα and anti-phospho-Smad3 antibodies for immunoblot analysis were purchased from Cell Signaling Technology (Beverly, MA). The cDNA encoding rat clusterin was purchased from Benebiosis (Seoul, Korea).
8
Collagen I Immunostaining in Cardiac Fibroblasts
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Immunocytochemistry was performed as described previously15 (link). Rat neonatal cardiac fibroblasts were seeded and serum starved overnight. The cells were pretreated with TGF-β1 (5 ng/mL) for 3 h and then with gallic acid (100 μM) for further 9 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 3% normal goat serum, and incubated overnight with anti-collagen type I antibody (1:200, Abcam, Cambridge, UK) at 4 °C. They were then probed with goat anti-rabbit IgG secondary antibody, Alexa Fluor 568 conjugate (1:400, Invitrogen). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear staining. The cells were then observed under a fluorescence microscope (Nikon Eclipse 80i; Nikon Corp., Tokyo, Japan). To compare the fluorescence intensity of collagen stain between the groups, the normalized mean intensity was calculated by an automated “measure tool” using the NIS Elements AR 3.0 program (Nikon, Japan).
9
Collagen Type I Immunostaining Protocol
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After standard histological processing procedures, immunostaining was performed using collagen type I antibody. The sections were deparaffinized in xylene and rehydrated with graded alcohol. Antigen retrieval was performed using 10 mM citrate-phosphate buffer (pH 6.0), the sections were subsequently blocked with 3% goat serum for 1 h, incubated with anti-collagen type I antibody (1:100, Abcam) at 4°C overnight, and incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (1:200, Invitrogen) for 1 h. Sudan black B solution was used to reduce autofluorescence. The sections were counterstained with DAPI and images were acquired using fluorescence microscopy (Nikon Eclipse 80i, Tokyo, Japan).
10
Immunohistochemical Analysis of Choroidal Flat Mounts
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