Indirect immunofluorescence staining was performed to detect the expression of collagen I and collagen III in the paraffin sections of LAA tissues. Tissue sections were blocked with normal bovine serum albumin (Zhongshan Goldenbridge Biotechnology, China) for 30 min, and then, the sections were incubated with polyclonal rabbit anti-human collagen I antibody (1 : 50, ab34710, Abcam) or monoclonal mouse anti-human collagen III antibody (1 : 200, ab6310, Abcam) overnight at 4°C. After washing with PBS solution with 0.1% polysorbate, the sections were incubated with the corresponding secondary antibodies at 37°C for 1 h. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1 : 100, Zhongshan Goldenbridge Biotechnology) was used as the secondary antibody for collagen I, and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1 : 100, Zhongshan Goldenbridge Biotechnology) was used as the secondary antibody for collagen III. Finally, the sections were washed with PBS solution thrice and counterstained with 4′,6-diamidino-2-phenylindole (1 : 100) for 1 h at room temperature. Negative controls were conducted without incubation with primary antibodies.
Ab6310
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab6310 is a mouse monoclonal antibody produced for use as a control in immunoassays. The antibody is purified from tissue culture supernatant.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (8 mentions)
Ab6308, by Abcam (4 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab7817, by Abcam (2 mentions)
Ab60178, by Abcam (2 mentions)
Col3a1, by Abcam (2 mentions)
Sc 8784 r, by Abcam (2 mentions)
Gapdh 6c5 sc 32233, by Santa Cruz Biotechnology (2 mentions)
Col1a1, by Abcam (2 mentions)
Ab150113, by Abcam (2 mentions)
Ab14041, by Abcam (2 mentions)
Ab88147, by Abcam (2 mentions)
Fluorescein isothiocyanate conjugated goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab270993, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dylight 594 conjugated goat anti mouse antibody, by Abcam (1 mentions)
Ab21610, by Abcam (1 mentions)
Spinning disc confocal microscope, by Zeiss (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Antifade mounting medium with dapi h 1200, by Vector Laboratories (1 mentions)
30 protocols using ab6310
1
Indirect Immunofluorescence for Collagen I and III
2
Histological Analysis of Supraspinatus Tendon
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The samples used for histological analysis were routinely fixed in 10% neutral buffered formalin, decalcified with 10% EDTA, embedded in paraffin, and cut into 4 μm-thick sections.
To obtain an overview of the supraspinatus tendon, the sections were stained with H&E. To detect the specific expression levels of type I collagen and type III collagen, the sections were stained with collagen I antibody (1:500, Abcam, ab270993) and collagen III antibody (1:400, Abcam, ab6310).
To obtain an overview of the supraspinatus tendon, the sections were stained with H&E. To detect the specific expression levels of type I collagen and type III collagen, the sections were stained with collagen I antibody (1:500, Abcam, ab270993) and collagen III antibody (1:400, Abcam, ab6310).
3
Quantifying Extracellular Matrix Proteins in UV-Damaged Fibroblasts
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Hs68 fibroblasts were seeded at a density of 2 × 105 cells/6 cm dish. After exposure to UVA radiation, cells were cultured in DMEM with or without adiponectin (10 μg/mL) for 24 h. After removing the supernatant, cells were fixed with 10% formaldehyde (30 min, RT), treated with 0.1% Nonidet P-40 (10 min, RT) and blocked with 2% bovine serum albumin (30 min, RT). Then, cells were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-collagen type I (ab 34710, 1:500, Abcam, Cambridge, UK), mouse anti-collagen type III (ab6310, 1:1000, Abcam), and rabbit anti-elastin (ab21610, 1:200, Abcam). After 2–3 rinses in PBS, cells were incubated for 30 min with secondary antibodies (DyLight 594 conjugated goat anti-mouse antibody, 1:50, Abcam or fluorescein (FITC-conjugated goat anti-rabbit antibody, 1:200, Jackson ImmunoResearch, West Grove, PA, USA). Finally, Samples were washed with PBS, stained with DAPI (4′-6-diamidino-2-phenylindole, Sigma-Aldrich, St. Louis, MO, USA), and imaged using a fluorescence microscope (Eclipse Ti-E, Nikon Cooperation) with CCD camera system (SPOT RT3, Diagnostic instruments Inc.). For quantitative analysis of elastin, type I and III collagen in Hs68 fibroblasts, we used ImageJ software to measure the fluorescence intensity in 10 images obtained with equal acquisition parameters.
4
Immunofluorescent Staining of Skin Sections
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Skin sections underwent deparaffinization and were permeabilized with 0.5% Triton X-100 in PBS for 5 min. They were next incubated with blocking buffer (5% normal donkey serum, 0.2% Triton-X in PBS) prior to primary antibody incubation at 4 °C overnight with the following antibodies diluted in blocking buffer: anti-TGF-β(1:400, MAB240-SP, R&D system, MN, US), anti-Collagen I (1:200, ab24821, Abcam, Cambridge, UK), and anti-Collagen III (1:400, ab6310, Abcam). After three 30-min washes with PBS + 0.05% Triton-X, samples were stained for 1 h at room temperature with fluorescent secondary antibodies (ThermoFisher Scientific, MA, USA) followed by two washes with PBS. The slides were mounted with Antifade Mounting Medium with DAPI (h-1200, Vector laboratories, CA, USA) and viewed under a spinning-disc confocal microscope (Carl Zeiss).
5
Western Blot Analysis of Muscle Collagens
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Protein levels of soleus muscle of CC and CT samples were analyzed by Western blotting using antibodies specific for collagen I (1:100) (sc-25974, Santa Cruz) and collagen III (1:5000) (ab6310, Abcam). Protein levels were normalized by the endogenous β-actin (1:1000) (sc-81178, Santa Cruz). Muscle protein was extracted using Tris-Triton buffer (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) containing Protease Inhibitor Cocktail (Sigma- Aldrich, USA) and quantified by the Bradford method72 (link). Samples were separated on a polyacrylamide gel and then transferred to a nitrocellulose membrane. After blockage, membranes were incubated with the primary antibody. Membrane was washed with TBS-T and incubated with secondary peroxidase-conjugated antibody (1:2500). Super Signal® West Pico Chemiluminescent Substrate (Pierce Protein Research Products, Rockford, USA) was used to detect bound antibodies.
6
Kidney Fibrosis Immunohistochemistry Protocol
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Staining was performed on 2-μm-thick paraffin kidney sections that were pretreated with a heat-based antigen retrieval approach. The primary antibodies used were Col3a (Abcam, #ab-6310), LOX (Abcam, #ab-60178), α-SMA (a specific marker of myofibroblasts) (Abcam, #ab-7817), Pan Cytokeratin (a specific marker of epithelial cell) (Abcam, #ab-7753) and CTR1 (Abcam, #ab-129067). The sections were incubated with the primary antibodies at 4 °C for 1–2 nights, followed by incubation with biotin-labeled secondary antibodies and horseradish peroxidase (HRP)-labeled tertiary antibodies (DAKO). The sections were developed using a 3,3′-diaminobenzidine (DAB) kit (Vector, #SK4100), counterstained with hematoxylin, dehydrated, and examined under a light microscope. For colocalization immunofluorescence staining, the slides were incubated with Alexa Fluor conjugated secondary antibody (Invitrogen, #Alexa Fluor 568-A11011, Alexa Fluor 488- A11029) for 2 h at room temperature. Zeiss confocal microscope was used to observe the labeled slides.
7
Quantitative Analysis of Extracellular Matrix Proteins
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Each section was incubated overnight at 4 °C with primary antibodies targeting Type I procollagen (ab64409, rat mAb, Abcam, Cambridge, UK, cross-reacts with human), Type I collagen (600–401-103, rabbit pAb, Rockland, Limerick, PA, cross-reacts with bovine and human), Type III collagen (ab6310, FH-7A, mouse mAb, Abcam, Cambridge, UK, cross-reacts with rat and human), Type V collagen (AM10159PU-N, V13F6, mouse mAb, Acris, San Diego, CA, cross-reacts with human), keratin-14 (20R-CP002, guinea pig pAb, Fitzgerald Industries International, Acton, MA) and PDGFRbeta (MAB1263, mouse mAb, R&D Systems, Minneapolis, MN). Primary antibodies were detected using Alexa488- or Alexa594-conjugated secondary antibody (Thermo Fisher Scientific). Sections were examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and images were captured with a DP72 controller digital camera (Olympus). The staining intensity in 6 randomly selected microscopic fields was quantified by using WINROOF2015 image-analyzing software (Mitani, Fukui, Japan, https://www.mitani-visual.jp/products/image_analys_ismeasurement/winroof/ ), as described previously10 (link).
8
Cardiac Protein Expression Analysis
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The protein lysate was added to the myocardial tissue placed on ice for 30 min and centrifuged to extract protein. The protein concentration was determined by BCA method. The protein was separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membrane was incubated with 5% skimmed milk powder at room temperature for 2 hours. Primary antibodies including collagen I antibody (dilution 1 : 1000) (#ab255809, Abcam), collagen III antibody (dilution 1 : 500) (#ab6310, Abcam), and β-actin antibody (dilution 1 : 5000) (#20536-1-AP, Proteintech Group, Inc., USA) were added and incubated at 4°C overnight. Then, the membrane was incubated with secondary antibody (dilution 1 : 5000) labelled by horseradish peroxidase (HRP) (#SA00001-2, Proteintech Group, Inc., USA) at room temperature for 2 hours. After the ECL luminescent reagent was coloured, the automatic gel imaging system was performed by exposure imaging. Image lab software was used to detect the expression of protein bands, and the relative expression of target protein was calculated with β-actin as the internal reference.
9
Western Blot Analysis of Collagen and Lysil Oxidase
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Protein expression of type I (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, col1a1, sc-8784-r) and type III collagen (Abcam, Cambridge, UK, col3a1, ab6310) and lysil oxidase (Abcam, LOX1, ab60178) was analyzed by Western blot as previously described [50 (link), 51 (link)]. Samples were separated on polyacrylamide gel and then transferred to a nitrocellulose membrane. After blockage, the membrane was incubated with the primary antibodies overnight at 4 °C. The membrane was then washed with PBS and Tween 20 and incubated with secondary peroxidase-conjugated antibodies (Santa Cuz Biotechnology, anti-mouse, sc-2005, and anti-rabbit, sc-2004) for 90 min at room temperature. ECL western blotting substrate (Pierce Protein Research Products, Rockford, USA) was used to detect bound antibodies. The membrane was then stripped (Restore Western Blot Stripping Buffer, Pierce Protein Research Products, Rockford, USA) to remove antibodies. After blockage, membrane was incubated with anti-GAPDH antibody (Santa Cruz Biotechnology, GAPDH 6C5 sc-32233). Protein levels were normalized to GAPDH.
10
Immunofluorescence Analysis of Periostin and Collagen III
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