Western Blot analysis was performed following standard protocols as previously described43 (link). Lung tissues were homogenized in 62.5 mM Tris Buffer, and then centrifuged at 12000 rpm for 10 min at 4 °C, and soluble supernatants were taken as whole tissue lysates. Equal amounts of protein mixed with sample buffer were separated on 10% SDS-PAGE gels, and the separated proteins were transferred onto a polyvinylidene fluoride (PVDF) transfer membrane at a constant voltage of 100 V for 100 min in ice-water mixture. The membranes were blocked with fat-free milk at room temperature for 1 h and incubated at 4 °C overnight with primary antibodies. The primary antibodies used in this study were anti-mouse FSTL1 (R&D system), anti-type I collagen (Abcam), α-SMA (sigma), NLRP3 and caspase-1 (Santa Cruz), β-tubulin (proteintech). After the HRP-conjugated secondary antibodies were incubated, protein signals were detected using the enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA). All experiments were repeated in triplicate.
Anti collagen type 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-collagen type I is a laboratory reagent used to detect and quantify collagen type I, a major structural protein found in various tissues, including bone, skin, and tendon. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by Abcam (3 mentions)
Anti α sma, by Abcam (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Anti fibronectin, by Abcam (3 mentions)
Anti smad3, by Cell Signaling Technology (2 mentions)
Anti p65, by Cell Signaling Technology (2 mentions)
Anti rig 1, by Proteintech (2 mentions)
Methyl green nuclear stain, by Vector Laboratories (2 mentions)
Poly l lysine coated slides, by Polysciences (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti α smooth muscle actin α sma, by Abcam (2 mentions)
Anti vimentin, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti laminin, by Merck Group (2 mentions)
Anti collagen type 3, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
β tubulin, by Proteintech (1 mentions)
α sma, by Merck Group (1 mentions)
Axiocam mrc5 digital camera, by Zeiss (1 mentions)
31 protocols using anti collagen type 1
1
Western Blot Analysis of Lung Tissue
2
Immunostaining for Profibrotic and Cell Proliferation Markers
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Formalin-fixed tissue was embedded in paraffin using standard procedure and histological sections were immunostained by standard avidin-biotin-peroxidase methodology (Vectastain ABC Kit, Vector Laboratories) using diaminobenzidine (DAB) as the chromogen. Profibrotic and cell proliferation status were assessed using antibodies anti-type I collagen (1:200, Abcam), anti-fibroblast-specific protein 1 (FSP-1) (1:800, Dako) and anti-KI-67 (1:1,000, Abcam), respectively. Digital microphotographs of renal cortex were obtained at 40-fold magnification using a Carl Zeiss Axioskop 40 microscope (Carl Zeiss Microscopy) and AxioCam MRc5 digital camera (Carl Zeiss Microscopy). The area stained positive for type I collagen, FSP-1 and KI-67 was quantified as percentage of total cross-sectional area using Axiovision software (Carl Zeiss Microscopy).
3
Western Blot Analysis of Fibrosis Markers
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Western blot was performed as previously described [19 (link)]. Membranes were incubated with anti-p-Smad3 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-Smad3 (1:1,000; Cell Signaling Technology), anti-PAI-1 (1:1,000; BD Biosciences), anti-α-SMA (1:1,000; Sigma), and anti-type I collagen (1:1,000; Abcam) polyclonal antibodies at 4℃ with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (Santa Cruz) using an Enhanced Chemiluminescence Western Blotting Detection System, in accordance with the manufacturer's instructions (Millipore, Billerica, MA, USA) [19 (link)]. The membrane was reblotted with anti-β-tubulin antibody (Applied Biological Materials Inc., Richmond, BC, Canada) to verify equal protein loading in each lane. Densitometric measurements of the bands were performed using UN-SCAN-IT digitizing software (Silk Scientific Corp., Orem, UT, USA).
4
Immunohistochemical Analysis of Key Proteins
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Immunohistochemical staining was performed as described [25 (link)]. The primary antibodies including anti-p65 (Cell Signaling Technology), anti-Ki67 (Abcam), anti-RIG-I (Proteintech, Sigma), and anti-type I collagen (Abcam) were used in immunohistochemical analyses.
5
Immunohistochemical and Histochemical Analysis of Cartilage
6
Western Blot and Immunohistochemistry Analysis
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Antibodies used for Western blot analysis were anti-rat α tubulin (Cell signaling Technology Japan, Tokyo, Japan) and anti-type I collagen (abbreviated as Col 1, abcam, Cambridge, UK). Anti-β1 and γ1 subunits of laminin (Lam b1 and Lam c1), and anti-fibronectin (FN1) were purchased from Santa Cruz Biotechnology (CA, USA). HRP-conjugated anti-rabbit IgG as secondary antibody and ECL plus Western blotting detection system (GE Healthcare, UK) were used for enhancing the signals. Antibodies used for immunohistochemistry were anti-Col 1 (Gentaur Molecular Products, Brussels, Belgium), anti-Lam (Affimetry BioReagents, CO, US), anti-FN1 (Affimetry BioReagents), and Alexa Fluor 488-conjugated secondary antibody (abcam). All other chemicals were of highest grade of purity commercially available.
7
Immunohistochemistry of 3D Dermis Models
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Immunohistochemistry was carried out using cross-sectioned tissues as previously described [33 (link)]. Briefly, Aronia extract-treated 3D dermis models were dehydrated 5 min and then fixed in 4% paraformaldehyde solution (Biosesang, Republic of Korea). Fixed tissues were transferred to frozen section compound (Leica Biosystems, Deutschland) to generate cryomolds. A 20 μm thick sectioned tissue was obtained using a cryotome (CM1860, Leica Biosystems). The cross-sectioned tissues were stained with anti-type I collagen (Abcam, UK) with Alexa® 488-conjugated goat anti-rabbit IgG (Invitrogen, CA, USA). To visualize cell nuclei in tissues, Hoechst 33342 (Invitrogen) staining was applied.
8
Western Blot Analysis of Key Signaling Proteins
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Total tissue or cellular lysate preparation and western blot analysis were performed as described previously [23 (link)]. The primary antibodies in the study are as follows. Anti-type I collagen, anti-α-smooth muscle actin (α-SMA), anti-RIG-I, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Abcam (Cambridge, MA, USA). Anti-IL-6, anti-IL-1β, and anti-c-Myc were obtained from Santa Cruz (Dallas, TX, USA). anti-c-Myc, anti-phospho-Smad3, anti-Smad3, anti-TGF-β, anti-phospho-p65, and anti-p65 were obtained from Cell Signaling Technology (Beverly, MA, USA). anti-RIG-I, Anti-type I collagen, anti-c-Myc, anti-TGF-β, and anti-fibronectin were obtained from Proteintech (Wuhan, China). Anti-IL-6 was obtained from GeneTex (Irvine, CA, USA). After 4 °C overnight incubation, the membranes were incubated with secondary antibodies from Beyotime. Protein bands were visualized by ECL system (AmerSham Biosciences).
9
Protein Expression Analysis by Western Blot
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With the use of RIPA buffer, proteins were extracted. The protein samples were electrophoretically segregated using a 10% SDS-PAGE. Then, the protein samples were transferred onto PVDF membranes and the incubation was performed overnight at 4 °C. The antibodies for Western blotting were: anti-eukaryotic initiation factor 5A2 (EIF5A2) (Abcam), anti-α-smooth muscle actin (α-SMA) (Abcam), anti-fibronectin (FN) (Abcam), anti-type I collagen (Abcam), anti-β-actin (Abcam), anti-CD9 (Abcam), anti-Tumor Susceptibility Gene 101 (TSG101) (Abcam), anti-E-cadherin (E-cad) and anti-desmin antibodies, at a dilution of 1:1000. Next, the second antibody (HRP-conjugated Affinipure goat anti-rabbit IgG; 1:5000 dilution; proteintech) was treated for 1 h at room temperature. Bands were visualized using an ECL chemiluminescent agent (Beyotime). An automatic chemical luminous imaging analysis system was used for capturing images. Quantification of protein expression was via ImageJ software (NIH ImageJ 1.47). β-actin was used as an internal reference to normalize protein expression.
10
Chondrocyte Characterization and Quantification
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After depolymerization of the alginate beads, cells from two patients were pelleted, rinsed with phosphate-buffered saline, resuspended in phosphate-buffered saline, placed on poly-l lysine–coated slides (Polysciences, Inc., Warrington, PA) and allowed to dry overnight. Histochemical localization of GAG was performed as previously described.23 (link)Single and clustered chondrocyte cell numbers was determined by counting eight random fields of view of for each oxygen tension assayed.
Immunohistochemistry was performed with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. Cells from two patients were prepared as described previously and probed with one of three antibodies: anti–type I collagen (7 μg/mL; no. ab6308; Abcam, Burlingame, CA), anti–type II collagen (0.5 μg/mL; no. 7005; Chrondrex, Inc., Redmond, WA), or a mouse nonspecific IgG (7 μg/mL; no. ab37355, Abcam) used as a negative control. Sections were counterstained with methyl green nuclear stain (Vector Laboratories). Samples were documented by photomicroscopy.
Immunohistochemistry was performed with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. Cells from two patients were prepared as described previously and probed with one of three antibodies: anti–type I collagen (7 μg/mL; no. ab6308; Abcam, Burlingame, CA), anti–type II collagen (0.5 μg/mL; no. 7005; Chrondrex, Inc., Redmond, WA), or a mouse nonspecific IgG (7 μg/mL; no. ab37355, Abcam) used as a negative control. Sections were counterstained with methyl green nuclear stain (Vector Laboratories). Samples were documented by photomicroscopy.
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