Fresh and glutaraldehyde fixed pericardial samples were embedded in Optimal Cutting Temperature material and cryosectioned (8 μm). Sections were air dried, fixed with acetone for 10 min and stored at −80 °C until used. Fresh and glutaraldehyde fixed samples were also fixed in formalin and paraffin embed for Masson’s Trichrome histology staining. Tissue sections were stained to detect the Gal antigen and collagens. Fresh and glutaraldehyde fixed tissues were stained with biotin-conjugated GSIB-4 (3 μg/ml) and murine monoclonal antibodies (2 μg/ml) to Collagen I (Anti-Collagen Type I, Sigma-Aldrich), III (Anti-Collagen Type III, Abcam, Cambridge, UK), and V (Anti-Collagen Type V, Abcam). Lectin and antibody solutions were produced in dPBS with 1% BSA and primary incubations were at 4 °C for 12 h. GSIB-4 binding was detected with HRP-conjugated streptavidin (Sigma-Aldrich) and mouse anti-collagen antibody binding was detected with biotin conjugated goat anti-mouse IgG (Sigma-Aldrich) and HRP-conjugated streptavidin. All sections were incubated with 3,3′-Diaminobenzidine (Sigma-Aldrich) and counter stained with haematoxylin.
Anti collagen type 3
Manufactured by Abcam
Sourced in United Kingdom, United States, Denmark, China
Anti-collagen type III is a laboratory reagent used for the detection and quantification of collagen type III in biological samples. It functions as an analytical tool for researchers studying the structure and composition of extracellular matrix proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti collagen type 1, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated streptavidin, by Merck Group (1 mentions)
Biotin conjugated goat anti mouse igg, by Merck Group (1 mentions)
Anti type 1 collagen, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Liquid dab plus substrate chromogen system kit, by Agilent Technologies (1 mentions)
Anti mlc 2v, by Proteintech (1 mentions)
Anti connexin 43, by Abcam (1 mentions)
Anti vimentin, by Agilent Technologies (1 mentions)
Anti sarcomeric alpha actinin α actinin, by Merck Group (1 mentions)
Anti cardiac troponin t ctnt, by Abcam (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Anti laminin, by Merck Group (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Anti mlc2a, by Synaptic Systems (1 mentions)
Lysis buffer, by Tiangen Biotech (1 mentions)
Biotinylated secondary antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Anti ctgf, by Abcam (1 mentions)
7 protocols using anti collagen type 3
1
Pericardial Tissue Histology and Glycan Analysis
2
Immunohistochemical Analysis of Collagen Types
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TL fasciae sections were immunolabeled with goat anti-collagen-type I or rabbit anti-collagen type III antibodies. Briefly, endogenous peroxidases were inactivated with PBS + 0.5% H2O2 for 10 min at room temperature; slides were then preincubated with blocking buffer (0.1% BSA in PBS) for 1 h at room temperature to decrease background staining. Sections were then incubated overnight at 4 °C with anti-collagen type I (1:400, SouthernBiotech, Birmingham, AL, USA) or anti-collagen type III (1:100, Abcam, Cambridge, UK) antibodies after washing with PBS; antigens were detected by incubation with secondary antibodies labeled with horseradish peroxidase (HRP) and 3,3′ –diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako, Santa Clara, CA, USA). Negative controls were performed by the omission of the primary antibody, confirming the specificity of the immunostaining.
3
Immunofluorescence Characterization of hiPSC-Derived Cardiomyocytes
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hiPSC-CMs and 3D-hiPSC-CT were fixed with 4% paraformaldehyde. hiPSC-CMs and 3D-hiPSC-CT were labeled by primary antibodies such as anti-cardiac troponin T (cTnT, 1:200 dilution; Abcam, Cambridge, UK), anti-sarcomeric alpha actinin (α-actinin, 1:400; Sigma), anti-vimentin (1:100; Dako, Glostrup, Denmark), anti-connexin43 (1:100; Abcam), anti-MLC2a (1:100; Synaptic Systems GmbH), anti-MLC2v (1:200; Proteintech, Rosemont) anti-fibronectin (1:200; Abcam), anti-laminin (1:30; Sigma), anti-collagen type I (1:100; Abcam), or anti-collagen type III (1:100; Abcam), followed by secondary antibodies such as AlexaFluor488 or AlexaFluor555 conjugated goat or donkey anti-mouse or anti-rabbit (ThermoFisher Scientific). Nuclei were counterstained with Hoechst33342 (Dojindo, Kumamoto, Japan) and assessed using confocal microscope (FLUOVIEW FV10i; Olympus, Tokyo, Japan).
4
Protein Expression Analysis in Lung Tissue
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Western blot analysis was employed to detect the protein expression level of TGF-β1, CTGF, and α-SMA in lung tissue, and collagen type III, α-SMA, and AdipoR1/R2 in human lung WI-38 fibroblasts. Whole lung tissue homogenate or WI-38 lung fibroblasts lysates were prepared via lysis buffer [TianGen Biotech (Beijing) Co., Ltd.]. The lung tissue homogenate or cell lysates were separated by electrophoresis on 10% SDS-PAGE. Gels were transferred to a nitrocellulose membrane, blocked in 5% nonfat dry milk diluted in Tris-buffered saline. Blots were incubated overnight with anti-TGF-β1, anti-CTGF, anti-α-SMA, anti-collagen type III, anti-AdipoR1, or anti-AdipoR2 antibodies (all from Abcam), at 4°C. Washed blots were incubated at room temperature for 60 minutes with a secondary biotinylated antibody (Zhongshan Golden Bridge Bio-technology, Beijing). After three 10 minute washings with TBST, membranes were analyzed by Western (Bio-Rad Laboratories Inc., Hercules, CA). Sample loadings were normalized by Western blot analysis with anti-GAPDH (Abcam).
5
Selenium Nanoparticle Modulation of Renal Injury
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HK-2 cells were cultured in six-well plates. Two wells were treated as one treatment group and divided into three groups (n=2 wells): control + PBS group, H/R + PBS group, and H/R + Se@SiO2 group. HK-2 cells and renal tissue from the outer medulla area were lysed and denatured at 100°C for 5 minutes in SDS buffer and separated by 10% PAGE. Proteins were then transferred onto 0.45-µm polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), blocked for 1 hour with 5% dried nonfat skimmed milk powder in TBS-Tween-20 (TBS containing 0.1% Tween 20), and probed with appropriate antibodies. The following primary antibodies were used: monoclonal rabbit antibodies anti-phospho-NF-κB (1:1,000 dilution; Cell Signaling Technology), anti-NLRP3 (1:1,000 dilution; Cell Signaling Technology), anti-collagen type I, anti-collagen type III (1:1,000 dilution; Abcam), anti-NGAL (1:1,000 dilution; Abcam), and caspase-1 (1:1,000 dilution; Proteintech, Wuhan, China) and monoclonal rabbit antibodies anti-glyceraldehyde-3-phosphate dehydrogenase and anti-β-actin (1:2,000 dilution; Proteintech). Positive protein binding was visualized by a horseradish peroxidase-conjugated secondary antibody followed by the use of an enhanced chemiluminescence kit (New Cell Molecular Biotech, Suzhou, China).
6
Multiparametric Tissue Immunohistochemistry
7
Integrin-Mediated Fibroblast Response to EDA-FN
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LPS from Escherichia coli serotype 0111:B4 and cFN from skin fibroblasts were from Sigma-Aldrich. TAK-242, TLR1/2 agonist Pam3Csk4, and TLR9 agonist CpG ODNs were purchased from Invivogen. pFN was from BD Bioscience. The following antibodies were used: anti-FN was from Chemicon (Merck-Millipore), and anti–EDA FN (FN-3E2), anti-FN (FN IST-4), and anti–β-actin were all from Sigma-Aldrich. Anti–EDA FN (IST-9) and anti–type III collagen were from Abcam. Anti–β3 integrin, anti-TLR4 (clone 25 and M-300), and anti-α4 (clone C19) were from Santa Cruz Biotechnology. Anti-mouse CD49d (α4 integrin) with blocking function was from BioLegend (clone 9C10). Anti–phospho-ERK 1/2, anti-ERK 1/2, anti–phospho–STAT-5, anti–STAT-5, anti–phospho p65 (NF-κB), and p65 (NF-κB) were purchased from Cell Signaling Technologies. EDA+ and EDA− recombinant peptides were produced as previously described (Losino et al., 2013 (link)). All the experiments were performed with the same batch for each peptide.
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