Knee joints were harvested and fixed in 10% neutral buffered formalin for 3 days, decalcified in Formic Acid Bone Decalcifier (Immunocal, Decal Chemical Corp.) for 7–10 days, paraffin processed, and embedded for sectioning. Tissues were sectioned at 5 µm and stained with ABH/OG. IHC analyses were performed on sections using traditional antigen retrieval and colorimetric development methodologies with the following primary antibodies: SOX9 (Santa Cruz), COL2A1 (Thermo Scientific), COL10A1 (Quartett), COL1A1 (Abcam), COL3A1 (Abcam), MMP-13 (Thermo Scientific), IL6 (Abcam), and phosphorylated STAT3 (Cell Signaling). TUNEL cell death assay was performed on sections using the in situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer’s instructions. IF analysis and β-galactosidase staining was performed on frozen sections. Knee joints were harvested and fixed in 4% paraformaldehyde (PFA) for 2 hours at 4 °C and decalcified with 14% EDTA at 4 °C for 10 days. Tissues were washed in sucrose gradient, embedded with Tissue-TEK OCT medium, snap frozen in liquid nitrogen and sectioned at 10 µm using a Lecia CM 1850 cryotome. The NOTCH1 primary antibody (Santa Cruz) was used for IF analysis. Beta-galactosidase staining was performed as previously described (63 (link)).
Col3a1
Manufactured by Abcam
Sourced in United Kingdom
COL3A1 is a protein-coding gene that provides instructions for the production of type III collagen, a structural component of various connective tissues in the body. The protein plays a crucial role in the formation and maintenance of these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Col1a1, by Abcam (3 mentions)
Gapdh 6c5 sc 32233, by Santa Cruz Biotechnology (2 mentions)
Sc 8784 r, by Abcam (2 mentions)
Ab6310, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Col2a1, by Thermo Fisher Scientific (1 mentions)
Cm1850 cryotome, by Leica (1 mentions)
Mmp 13, by Thermo Fisher Scientific (1 mentions)
Phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Notch1 primary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab60178, by Abcam (1 mentions)
Fibronectin, by BD (1 mentions)
Actr iib, by Santa Cruz Biotechnology (1 mentions)
Keratin 8, by Abcam (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Smad7, by Santa Cruz Biotechnology (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
6 protocols using col3a1
1
Histological Analysis of Knee Joints
2
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.
3
Western Blot Analysis of EMT Markers
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Whole-cell protein extracts were obtained with radio-immunoprecipitation assay buffer following standard protocols. The protein extracts were denatured and 40–50 μg of each sample were separated on 12% SDS–PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk (LabScientific, Inc.) in TBS-Tween buffer, the membranes were probed with antibodies against E-cadherin (1:250, Santa Cruz), N-cadherin (1:250, Santa Cruz), Smad7 (1:200, Santa Cruz), ACTR-IIA (1:500, Santa Cruz), ACTR-IIB (1:500, Santa Cruz), actin (1:1,000, Santa Cruz), keratin 8 (1:250, Epitomics), Col3A1 (1:200, Abcam), Smad2 (1:500, Cell Signaling), P-Smad2 (1:250, Cell Signaling), Smad3 (1:200, Cell Signaling), P-Smad3 (1:100, Cell Signaling) and fibronectin (1:1,000, BD Transduction Lab). Membranes were exposed using enhanced chemiluminescence (Roche) method following manufacturer’s instructions. Full scans of all western blots are included in Supplementary Fig. S10 .
4
Collagen and Lysyl Oxidase Expression
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The expression of lysil oxidase (Abcam, LOX1, ab60178) and Type I (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, col1a1, sc-8784-r) and Type III collagen (Abcam, Cambridge, UK, col3a1, ab6310) was assessed by Western blot [37 (link)]. After protein extraction, the samples were separated on polyacrylamide gel and transferred to a nitrocellulose membrane. Polyacrylamide concentration was 10% for Types I and III collagen, and 12% for lysyl oxidase. After blockade with milk, the membrane was incubated with primary antibodies overnight at 4 °C. Next, the membrane was 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. Bound antibodies were detected using ECL Western Blotting Substrate (Pierce Protein Research Products, Rockford, IL, USA). After stripping antibodies from the membrane (Restore Western Blot Stripping Buffer, Pierce Protein Research Products, Rockford, IL, USA), it was incubated with anti-GAPDH antibody (Santa Cruz Biotechnology, GAPDH 6C5, sc-32233). Protein levels were normalized to GAPDH.
5
Protein Expression Analysis Protocol
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Total proteins were extracted from tissues and cells after 72 h cultivation by using lysis buffer (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) and quantified by Pierce BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.) according to the manufacturer’s protocol. Equal amount of protein was separated on denaturing SDS gel and transferred to a PVDF membrane. The membrane was blocked with 10% skim milk and the incubated with primary antibodies overnight at 4°C, followed by incubation in appropriate horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The membrane was washed twice with PBS and blots were then visualized by ECL system (Bio–Rad Laboratories, Hercules, CA, U.S.A.). GAPDH was used as loading control. The specific primary antibodies were as follows: anti-COL1A1, COL3A1, Timp-1, MMP-3, Cyclin D1, c-fos (Abcam, U.S.A.), MEK1/2 and p-MEK1/2, ERK1/2 and p-ERK1/2 (Cell Signaling, Beverly, MA, U.S.A.).
6
Lung Tissue Protein Extraction and Western Blot Analysis
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Total proteins were extracted from lung tissues using the RIPA buffer (Solarbio, Beijing, China) in the presence of Protease Inhibitor Cocktail (Solarbio) and Protein Phosphatase Inhibitor (Solarbio), loaded on an SDS-PAGE gel, and electroblotted onto PVDF membrane. The primary antibodies were applied for overnight in 5% BSA at 4°C. WB were analyzed using ImageLab (Bio-Rad, Berkeley, CA). Primary antibodies: β-actin antibody (Abcam, Cambridge, UK); NF-κB p65 (Abcam); p-NF-κB p65 (Abcam); phospho-myosin phosphatase target subunit 1 (p-MYPT1; Abcam); MYPT1(Abcam); RhoA (Abcam); peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α; Abcam); nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4; Abcam); intercellular adhesion molecule (ICAM-1; Abcam); transforming growth factor-beta1 (TGF-β1; Abcam); COL3A1 (Abcam); COL1A2 (Abcam).
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