Proteins were extracted from kidney and brain nuclei tissue samples, and separated using 10% SDS–PAGE. The protein expression levels of RAS components, catalase, NOX2, MCP-1, IL-6, fibronectin, and collagen I in the kidney cortex and specific brain nuclei were determined with anti-AGT (IBL), anti-ACE (Abcam), anti-ACE2 (Abcam), anti-Mas receptor (Alomone Labs), anti-NOX2 (Santa Cruz Biotechnology), anti-catalase (Abcam), anti-MCP-1 (Abcam), anti-IL-6 (Abcam), anti-fibronectin (Sigma), anti-collagen I (BOSTER), and anti-β-actin (Cell Signaling Technology) primary antibodies. The membranes were then incubated with secondary antibodies for 1 h, washed, and analyzed.
Anti collagen 1
Manufactured by Boster Bio
Sourced in China
Anti-collagen I is a laboratory reagent used to detect and quantify collagen type I in biological samples. It functions as a specific antibody that binds to collagen type I, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibronectin, by Merck Group (3 mentions)
Anti collagen 3, by Boster Bio (3 mentions)
Anti cd44, by Boster Bio (2 mentions)
Anti cd63, by Abcam (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Anti ace2, by Abcam (1 mentions)
Anti ace, by Abcam (1 mentions)
Anti nox2, by Santa Cruz Biotechnology (1 mentions)
Anti il 6, by Abcam (1 mentions)
Anti mcp 1, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti catalase, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Fluorescence microscope, by Solarbio (1 mentions)
Anti kir2, by Abcam (1 mentions)
Anti α sma, by Boster Bio (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti foxo4, by Cell Signaling Technology (1 mentions)
Anti active β catenin, by Cell Signaling Technology (1 mentions)
Anti pdgfr β, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti collagen 1
1
Quantifying RAS-related Proteins in Kidney and Brain
2
Quantification of Collagen I and III in Atrial Tissue
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The atrial myocardium samples were weighed and treated with cold Radio-Immunoprecipitation Assay (RIPA) lysis buffer (1 ml/100 mg). The homogenized samples were centrifuged (12,000 rpm, 10 min, 4 °C) and a BCA protein assay kit (Yeason Biotech, Shanghai, China) was used to determine their protein concentrations. Loading buffer was added to diluted supernatants to adjust concentrations and volumes. Afterward, samples with equal protein content were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel, then transferred to a polyvinylidene difluoride membrane (Millipore, Boston, MA, USA). Next, the samples were blocked with blocking buffer containing 5% bovine serum albumin for 1 h at room temperature, followed by incubation with antibodies including anti-collagen I (Boster Biological Technology, Wuhan, China) and anti-collagen III (Boster Biological Technology, Wuhan, China), overnight at 4 °C, with anti-GAPDH used as an internal control. The membranes were rinsed 3 times for 10 min with TBST, followed by incubation with the secondary antibody for 1 h. The membranes were washed 3 times with TBST for 15 min. Then, the membranes were developed with chemiluminescence solution A and B mixed at a 1:1 ratio, along with the developing substrate. Image J software was used to calculate the relative optical density of the bands.
3
Immunofluorescence Assay for Kir2.1, α-SMA, Collagen I, and III
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After culturing and incubation for 24 h, the cells were washed with PBS three times (2 min each) and then fixed with paraformaldehyde for 10 min. After being washed with PBS three times (2 min each), the cell membrane was permeabilized with Triton X-100 for 1–3 min and then washed again with PBS three times (2 min each). After blocking with 10% BSA for 30 min, the antibodies were incubated with anti-Kir2.1 (1:100; Abcam), anti-α-SMA (1:100; Boster), anti-collagen I (1:100; Boster), or anti-collagen III (1:100; Boster) antibodies at 4°C overnight. After 30 min of rewarming the next day, the corresponding fluorescein-labelled antibody was added and incubated for 2 h at 37°C. PBS was used to wash the cells three times (10 min each). DAPI was used to stain the nuclei for 10 min, and then cells were observed under a fluorescence microscope (Solarbio, Beijing, China), and images were captured. ImageJ software (NIH, Bethesda, USA) was used for fluorescence image analysis.
4
Quantifying Collagen in Tissue Samples
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As we previously reported [15 (link)], the Masson triple staining and Western Blotting were done according to the protocols. For Masson staining, the sections were observed under a light microscope, and the image data were collected to calculate the volume fraction of collagen (CVF% = average collagen area/area of total field× 100).
For Western Blotting, the protein expression level of Collagen I and Collagen III were tested, with the antibodies including anti-collagen I (Boster Biological Technology, Wuhan, China) and anti-collagen III (Boster Biological Technology, Wuhan, China) adopted, and anti-GAPDH used as an internal control. Image J software was used to calculate the relative optical density of the bands.
For Western Blotting, the protein expression level of Collagen I and Collagen III were tested, with the antibodies including anti-collagen I (Boster Biological Technology, Wuhan, China) and anti-collagen III (Boster Biological Technology, Wuhan, China) adopted, and anti-GAPDH used as an internal control. Image J software was used to calculate the relative optical density of the bands.
5
Western Blot Analysis of Protein Expression
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Protein expression was analyzed by western blot analysis as described previously (Zhou et al., 2015 (link)). The following primary antibodies were used: anti‐N‐OPN (Abcam, Cat. ab181440, 1:1000), anti‐CD63 (Abcam, Cat. ab59479, 1:1000), anti‐OPN (Boster Biotechnology, Cat. PB0589, 1:1000), anti‐CD44 (Boster Biotechnology, Cat. A00052, 1:1000), anti‐α‐tubulin (Beijing Ray Antibody Biotech, Cat. RM2007, 1:5000), anti‐fibronectin (Sigma, Cat. F3648, 1:50000), anti‐α‐SMA (Abcam, Cat. ab5648, 1:1000), anti‐PDGFR‐β (Santa Cruz, Cat. sc‐374573, 1:1000), anti‐Collagen I (Boster Biotechnology, Cat. BA0325, 1:1000), anti‐Vimentin (Abcam, Cat. ab8978, 1:1000), anti‐PCNA (Abcam, Cat. ab29; 1:1000), anti‐active‐β‐catenin (Cell Signaling, Cat. #4270s, 1:1000), anti‐c‐Myc (Cell Signaling, Cat. #5605s, 1:1000), and anti‐Foxo4 (Cell Signaling, Cat. #9472s, 1:1000), anti‐TSG101 (Abcam, Cat. Ab83; 1:1000), anti‐CD81 (Boster Biotechnology, Cat. A01281‐2, 1:1000), anti‐Alix (Boster Biotechnology, Cat. BM5496, 1:1000), anti‐Flag (Boster Biotechnology, Cat. M30971, 1:1000).
6
Immunofluorescence Analysis of Kidney and Cell Markers
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Frozen sections of kidneys were fixed with 4% paraformalin‐fixing solution for 15 min at room temperature. NRK‐49F cells cultured on coverslips were fixed with cold methanol:acetone (1:1) for 15 min at room temperature, followed by blocking with 10% normal donkey serum in PBS. Slides were incubated with primary antibodies against anti‐N‐OPN (Abcam, Cat. ab181440, 1:50), anti‐OPN (Boster Biotechnology, Cat. PB0589, 1:50), anti‐CD63 (Abcam, Cat. ab59479, 1:50), anti‐Lotus Tetragonolobus Lectin (LTL) (VECTOR Laboratories, Cat. FL‐1321,1:400), anti‐Peanut Agglutinin (PNA) (VECTOR Laboratories, Cat. FL‐107, 1:400), anti‐Dolichos Biflorus Agglutinin (DBA) (VECTOR Laboratories, Cat. FL1031, 1:400), anti‐Collagen I (Boster Biotechnology, Cat. BA0325, 1:50), anti‐Vimentin (Abcam, Cat. ab8978, 1:50), anti‐fibronectin (Sigma, Cat. F3648; 1:100), anti‐CD44 (Boster Biotechnology, Cat. M00052‐3, 1:50) and anti‐β‐catenin (BD Biosciences, Cat. 610154, 1:150). After washing with TBS‐T, slides were incubated with Cy2 or Cy3‐conjugated donkey anti‐ or anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (Beyotime, Cat. C1006) according to the manufacturer's instructions. All images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).
7
Histological and Immunohistochemical Analysis of Lung Tissue
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The lung tissues were fixed in 4% paraformaldehyde 48 hours. After dehydration, tissues were embedded in paraffin, and 5-μm-thick paraffin sections were cut as slides for pathological staining like hematoxylin and eosin (H&E), Periodic Acid Schiff (PAS), and Masson’s trichrome.
Immunohistochemical staining was performed using standard procedures. After deparaffinization and hydration using xylene and sequentially decreasing concentrations of ethanol, the sections were repaired in antigen repair solution and treated with 3% hydrogen peroxide (H2O2) to remove endogenous peroxides. Then, the sections were blocked with goat serum for 1 hour. The samples were incubated with primary antibodies (anti-SPARC [1:200; HUABIO], anti-E-cadherin [1:200; Cell Signaling Technology], anti-N-cadherin [1:200; Cell Signaling Technology], anti-fibronectin [1:200; HUABIO], anti-collagen I [1:200; BOSTER], anti-collagen III [1:200; Abcam], anti-α-SMA [1:200; HUABIO]) overnight at 4°C, followed by HRP-labelled secondary antibody at 37°C for 30 minutes. Color development was performed with 3,3-diaminobenzidine solution, and the reaction was stopped with PBS.
Immunohistochemical staining was performed using standard procedures. After deparaffinization and hydration using xylene and sequentially decreasing concentrations of ethanol, the sections were repaired in antigen repair solution and treated with 3% hydrogen peroxide (H2O2) to remove endogenous peroxides. Then, the sections were blocked with goat serum for 1 hour. The samples were incubated with primary antibodies (anti-SPARC [1:200; HUABIO], anti-E-cadherin [1:200; Cell Signaling Technology], anti-N-cadherin [1:200; Cell Signaling Technology], anti-fibronectin [1:200; HUABIO], anti-collagen I [1:200; BOSTER], anti-collagen III [1:200; Abcam], anti-α-SMA [1:200; HUABIO]) overnight at 4°C, followed by HRP-labelled secondary antibody at 37°C for 30 minutes. Color development was performed with 3,3-diaminobenzidine solution, and the reaction was stopped with PBS.
8
Immunohistochemical Analysis of Myocardial Tissues
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Myocardial tissues were fixed with 4% paraformaldehyde, embedded in a paraffin block, and cut into 3-μm sections. After a series of deparaffinization and dehydration steps, endogenous peroxidase activity was blocked with 3% H2O2, and then freshly prepared 0.01 mol/l citrate buffer was used for antigen retrieval. After washing three times with PBS, the sections were blocked with 5% BSA for 30 min, and then incubated with the following primary antibodies: anti-α-SMA (1：200; MAXIM, Fujian, China), anti-Periostin (1：200; Proteintech), anti-Collagen I (1：200; Boster, Wuhan, China), anti-IL-1β (1：200; Bioworld Technology), anti-TNF-α (1：200; Bioworld Technology), and anti-TGF-β (1：300; Abcam) overnight at 4℃. Then the sections were incubated with biotin-la-beled goat anti-mouse/rabbit secondary antibodies at 37℃ for 30 min. DAB chromogenic solution was used for color development, and hematoxylin was used for counter staining. Immunohistochemical images were captured with a Panoramic Scanner MIDI (3DHISTECH, Budapest, Hungary).
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