Liver sections were stained with haematoxylin and eosin (H&E) for pathological evaluation, as described before (17 (link)). Ethanol-induced steatosis was quantified as the percentage of cells containing fatty droplets; five fields (200×) per liver section were examined (one 200× field area contains about 200 cells). Steatosis scoring was recorded as follows: 0, none; 1, <5%; 2, 5–33%; 3, 34–66%; and 4, >67%. Ethanol-induced necroinflammation was quantified as the number of clusters of 5 or more inflammatory cells per mm2; for this, five 200× fields per liver were examined (one 200× field area = 0.95 mm2). Necrosis scoring was recorded as follows: 0, none; 1, <2 foci per mm2; 2, 2–4 foci per mm2; 3, 5–10 foci per mm2; and 4, >10 foci per mm2. The pathologists were unaware of the treatment groups when evaluating the slides. Immunohistochemical staining (IHC) for 4-hydroxyl-nonenal (4-HNE), 3-nitro-tyrosine (3-NT), collagen I, and alpha smooth muscle actin (α-SMA) was performed by using anti-4-HNE, anti-3-NT, anti-collagen I, and anti-α-SMA antibodies (Millipore), followed by the use of a Broad Spectrum (AEC) Histostain-Plus kit (Invitrogen). No staining was observed in the absence of the primary antibody.
Anti collagen 1
Manufactured by Merck Group
Sourced in United States, Germany
Anti-Collagen I is a laboratory reagent used for the detection and quantification of Collagen I, a major structural protein found in connective tissues. It functions as an antibody that binds specifically to Collagen I, enabling researchers to analyze its presence and levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti collagen 3, by Merck Group (3 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Anti phospho smad3, by Cell Signaling Technology (2 mentions)
Anti fibronectin, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Histostain plus aec kit, by Thermo Fisher Scientific (1 mentions)
Anti smad2 3, by BD (1 mentions)
Hrp conjugated donkey anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated sheep anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti erk1, by Cell Signaling Technology (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Bio-Rad (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
14 protocols using anti collagen 1
1
Quantitative Assessment of Ethanol-Induced Liver Pathology
2
Signaling Pathways Activated by Growth Factors
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Prior to treatment with TGF-β1 (4 ng/mL, R&D), BMP-2 (50 ng/mL, R&D), FGF-2 (20 ng/mL, Stemgent) or SB431542 (10 µM, Tocris) for the indicated time, cells were starved overnight in α-MEM containing 0.2% FCS. After stimulation and two washes with ice cold PBS, cells were harvested in RIPA lysis buffer (Millipore, Billerica, MA, USA) with protease and phosphatase inhibitors. The protein concentrations of the cell lysates were determined by the Bradford method (Bio-Rad, Hercules, CA, USA). Primary antibodies included anti-phospho-Erk, anti-phospho-p38, anti-Erk1, anti-p38, and anti-phospho-Smad3 (1:2000, Cell Signaling, Danvers, MA, USA); anti-Smad2/3 (1:2000, BD Biosciences, San Jose, CA, USA); anti-BMP type I receptor, anti-BMP type II receptor, anti-TGF-β type I receptor, and anti-TGF-β type II receptor (1:500, Santa Cruz, Santa Cruz, CA, USA); anti-collagen I (Millipore); and anti-β-actin (1:5000, Sigma-Aldrich). Secondary antibodies included horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG (1:5000, Cell Signaling).
3
Histological and Protein Analysis of Aorta
4
TGF-β1 Signaling Modulation Assay
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). HCQ sulfate was purchased from Thermo Fisher Scientific (Waltham, MA, USA). CQ phosphate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Compound C was purchased from Calbiochem (San Diego, CA, USA). Anti-phospho-AMPK, anti-AMPK, anti-LC3, anti-phospho-Smad2, and anti-phospho-Smad3 antibodies were from Cell Signaling Technology (Danvers, MA, USA), and anti-p27 and anti-Smad3 antibodies were from Santa Cruz Biotechnology. Anti-Cyclin D and anti-Collagen I were from Merck-Millipore (Temecula, CA, USA). anti-Collagen III was from Fitzgerald Industries International (Acton, MA, USA). Anti-tubulin was from Sigma-Aldrich (St. Louis, MO, USA).
5
Quantification of Collagen I and III in LV Tissue
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First, frozen LV tissue samples were embedded in Tissue‐Tek OCT (Sakura, Zoeterwoude, NL) and cut into 5 μm thick sections for subsequent immunohistological investigations. To this end, slides were incubated with the following antibodies: anti‐collagen I (dilution 1:350; Chemicon, Merck Millipore, Darmstadt, Germany) and anti‐collagen III (dilution 1:200; Calbiochem, Merck Millipore, Darmstadt, Germany) using the EnVision® method. After the respective staining, samples were investigated on a Leica DM2000 LED microscope (Leica Microsystems GmbH, Wetzlar, Germany) at 100× magnification, followed by digital image analysis via the Leica Application Suite version 4.4 (LAS V4.4). In general, specific epitopes of the stained structure are coloured red and the heart area (HA) was counterstained with Hemalum (blue). To distinguish between different types of fibrosis, LV samples were analysed including vessel and arteries (total LV area) and without vessels and arteries (interstitial fibrosis).
6
Immunohistochemical Analysis of Vascular and Immune Markers
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Immunohistochemistry was carried out on 5 µm‐thick cryosections using following antibodies: anti‐collagen I (Chemicon, Limburg/Lahn, Germany), anti‐collagen III (Merck Millipore, Darmstadt, Germany), anti‐CD68 (Abcam, Cambridge, U.K.), anti‐CD3 (Santa Cruz Biotechnology, Heidelberg, Germany) anti‐CD4 (BD Biosciences, San Jose, CA), and anti‐CD8a (BD Biosciences). Analysis of stained sections was made in a blinded fashion by digital image analysis on a Leica DMRB microscope (Leica Microsystems, Wetzlar, Germay) at ×200 magnification. Artery and arteriole density was measured using immunohistochemistry with an anti‐α‐smooth muscle actin (SMA) antibody (1:200, Abcam). Microscopy was performed on a Leica DM2000 light microscope. Arteries and arterioles were counted in 10 high power fields (hpf) per animal at a magnification of ×100. The density is presented as number of arteries or arterioles per hpf.
7
Immunohistological Staining for Cell Quantification
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As described previously,46 immunohistological stainings were performed using the indirect method by incubation with a specific primary antibody, subsequent incubation with an appropriate secondary antibody, and final visualization by a horseradish peroxidase reaction. Anti‐CD11b (Pharmingen, San Diego, CA, USA) followed by DAKO EO 468 (DAKO, Glostrup, Denmark) was used to quantify CD11b‐positive cells/heart area (HA). Anti‐intercellular adhesion molecule (anti‐ICAM)‐1 (Pharmingen, San Diego, CA, USA) in combination with Dianova #127‐035‐160 (Dianova, Hamburg, Germany), and anti‐collagen I (Chemicon, Billerica, MA, USA), anti‐collagen III (Calbiochem, Darmstadt, Germany), and anti‐nitrotyrosine (Sigma‐Aldrich, St. Luis, MO, USA) in combination with EnVision‐anti‐rabbit (DAKO, Glostrup, Denmark) was used to determine LV immune cell infiltration, fibrosis, and oxidative stress as AF or number of cells/HA.
8
Immunofluorescence Characterization of Cellular and Extracellular Matrix Components
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Both cellularized (with v-HCFs or a-HCFs) and decellularized samples were fixed in paraformaldehyde 4% in PBS (PFA, Alfa Aesar) for 15 min, washed with PBS, and cells were permeabilized with Triton X-100 (Sigma-Aldrich) 0.5% in PBS for 10 min. Samples were then blocked with bovine serum albumin (BSA, Sigma-Aldrich) 2% in PBS for 30 min, followed by staining with Phalloidin-Rhodamine (ThermoFisher) or primary and secondary antibodies, diluted in BSA 2% in PBS. Primary antibodies for fibroblasts staining were: Anti-Actin Smooth Muscle (α-SMA, Sigma Aldrich) and Anti-Discoidin Domain Receptor 2 (DDR2, ThermoFisher). Primary antibodies used for extracellular matrix protein detection were anti-Collagen I, anti-Collagen III, anti-Fibronectin, anti-Laminin, anti-Tenascin, (all purchased from Sigma-Aldrich), and anti-Collagen IV (Abcam). Secondary antibodies used were anti-mouse Alexa Fluor 555 and anti-rabbit Alexa Fluor 488 (both from ThermoFisher). Nuclei were counterstained with DAPI (Sigma-Aldrich). Samples were maintained in PBS during imaging by using Nikon Ti2-E fluorescence microscope (Nikon Instruments). Immunofluorescence experiments were performed in biological triplicate.
9
Western Blot Analysis of Osteogenic Markers
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Total protein was extracted from cells by radioimmunoprecipitation assay buffer containing protease inhibitors. The protein concentration was detected using a BCA protein assay (BioRad, USA). Then, equal amounts of protein were loaded and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with the following specific primary antibodies: anti-SOX5, anti-Collagen I, anti-Runx2, anti-Osterix, and anti-KLF4 (Sigma-Aldrich, USA), and anti-β-actin antibody (Sigma, USA). After incubation with a peroxidase-conjugated secondary antibodies (Sigma) for 2 h at room temperature, the target proteins were visualized using enhanced chemiluminescence reagents (Pierce, USA) on Image Reader LAS-3000 Fujifilm. The density of the bands on the membrane were quantified with Image J software. β-actin was used as an internal control.
10
Immunophenotyping of Aortic Cell Populations
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