For Western blot, whole-cell extracts were prepared from HDMECs using lysis buffer with the following composition: 1% Triton X-100, 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 3 mmol/L MgCl2, 1 mmol/L CaCl2, proteinase inhibitor mixture (Roche), and 1 mmol/L phenylmethyl sulfonyl fluoride. Protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated overnight with primary antibodies, washed, and incubated for 1 hour with appropriate horseradish peroxidase-conjugated secondary antibody. After washing, visualization was performed by enhanced chemiluminescence (Pierce, Rockford, IL). The following primary antibodies were used: mouse anti-human FLI1 (BD Biosciences, Billerica, MA), rabbit anti-human FOXO3A (Abcam, Cambridge, MA), rabbit polyclonal SNAI1 (Santa Cruz, CA), goat anti-Collagen type I (Southern Biotech, Birmingham, AL) rabbit anti-human FOXO3A (p Ser253) (Novus Biologicals, Littleton, CO) at a dilution of 1:1,000 dilution, and a control mouse monoclonal anti β-actin (Sigma, St Louis, MO) at a dilution of 1:5,000. Proteins levels were quantified using Image J software.
Goat anti collagen type 1
Manufactured by Southern Biotech
Sourced in United States
Goat anti-collagen type I is a laboratory reagent used to detect and quantify the presence of collagen type I in various samples. It functions as a specific antibody that binds to collagen type I, enabling its identification and measurement through various immunological techniques.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase rabbit anti goat, by Jackson ImmunoResearch (4 mentions)
Hrp labeled goat anti rabbit igg, by Agilent Technologies (4 mentions)
Liquid dab plus substrate chromogen system kit, by Agilent Technologies (2 mentions)
Mouse anti α sma, by Merck Group (2 mentions)
Snai1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti human foxo3a, by Abcam (1 mentions)
Horseradish peroxidase hrp labeled rabbit anti mouse igg, by Agilent Technologies (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Bx53 microscope, by Olympus (1 mentions)
Rabbit antiadiponectin, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Rabbit anti α sma, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
A1 laser scanning confocal microscope, by Nikon (1 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Rabbit anti p16, by Santa Cruz Biotechnology (1 mentions)
Mouse anti cd81, by Santa Cruz Biotechnology (1 mentions)
9 protocols using goat anti collagen type 1
1
Protein Expression Analysis in HDMECs
2
Immunostaining of Collagen Types I and III
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After blocking of endogen peroxidase by 0.5% H2O2 in PBS for 10 min at room temperature and repeated washings in PBS, samples were pre-incubated with a blocking 179 buffer (0.1% BSA in PBS) for 60 min at room temperature, and then incubated in goat anti-collagen-Type I, (1:400, SouthernBiotech, Birmingham, AL, USA), or rabbit anti-collagen-III, N-terminal antibody (1:100, Abcam, Cambridge, UK), overnight at 4 °C. After repeated PBS washings, samples were maintained for 1 h in secondary antibody (Collagen Type I), peroxidase Rabbit Anti-Goat and (Collagen III) peroxidase-Goat Anti-Rabbit (1:300, Jackson Immu-noResearch Laboratories, Inc., West Grove, PA, USA), and then washed in PBS. The reaction was developed with 3,3′-diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako, Glostrup, Denmark). Negative controls were conducted by the omission of the primary antibody, confirming the specificity of the immunostaining.
3
Characterization of CAF Spheroids by Immunostaining
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Spheroid characterization was performed for expression of the CAF (cancer-associated fibroblast) biomarkers by immunostaining. Spheroids grown in microwells for 48 h were washed with PBS and subsequently embedded in Cryomatrix™, cut into 8-μm-thick sections and processed for immunostaining. Cryosections were fixed with acetone at room temperature (RT) for 15 min, rehydrated in PBS, and incubated with either mouse anti-α-SMA (Sigma-Aldrich, 1:400) or goat anti-collagen type I (Southern Biotech, 1:100) in PBS for 1 h at RT. Subsequently, sections were washed in PBS again and incubated with secondary antibody—horseradish peroxidase (HRP)–labeled rabbit anti-mouse IgG (DAKO, 1:100) or HRP-labeled goat anti-rabbit IgG (DAKO, 1:100) in PBS for 1 h. Sections were rewashed with PBS and finally incubated with a tertiary antibody—HRP-labeled goat anti-rabbit IgG (DAKO, 1:100) or HRP-labeled rabbit anti-goat IgG (DAKO, 1:100) in PBS for 1 h. AEC (3-amino-9-ethyl-carbazole) in Milli-Q water was applied for 20 min to develop peroxidase activity and resulting in red staining. Samples were subsequently counterstained with hematoxylin to visualize cell nuclei, washed in running tap water for 5 min, and mounted with Aquatex®. Imaging was performed using Nanozoomer-RS.
4
Immunohistochemical Analysis of Collagen Types
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After blocking of endogen peroxidase by 0.5% H2O2 in PBS for 10 min at room temperature and repeated washings in PBS, samples were pre-incubated with a blocking buffer (0.1% BSA in PBS) for 60 min at room temperature and then incubated in goat anti-collagen type I, (1:400, SouthernBiotech, Birmingham, AL, USA), or rabbit anti-collagen-III, N-terminal antibody (1:100, Abcam, Cambridge, UK), overnight at 4 °C. After repeated PBS washings, samples were maintained for 1 h in secondary antibody (collagen type I), peroxidase rabbit anti-goat and (collagen III) peroxidase goat anti-rabbit (1:300, Jackson ImmunoResearch Laboratories, Inc.), and then washed in PBS. The reaction was developed with 3,3′–diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako). Negative controls were conducted via omission of the primary antibody, confirming the specificity of the immunostaining (Appendix A Figure A1 B,C).
5
Immunostaining of Spheroid Cryosections
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Spheroids grown in microwells were washed with PBS and subsequently embedded in Cryomatrix™. The resulting spheroid array was cut into 8 μm-thick sections and processed for immunostaining. Cryosections were first fixed with acetone at room temperature (RT) for 15 min, rehydrated in PBS and incubated with either mouse anti-α-SMA (Sigma-Aldrich, 1:400), or goat anti-collagen type I (Southern Biotech, 1:100) in PBS for 1 h at RT. Subsequently, sections were washed again in PBS and incubated with secondary antibody-horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG (DAKO, 1:100) or HRPlabeled goat anti-rabbit IgG (DAKO, 1:100) in PBS for 1 h. Sections were washed again with PBS and finally incubated with a tertiary antibody -HRP-labeled goat anti-rabbit IgG (DAKO, 1:100) or HRP-labeled rabbit anti-goat IgG (DAKO, 1:100) in PBS for 1 h. The peroxidase activity was developed with 3-amino-9-ethyl-carbazole (AEC) in MilliQwater for 20 min. Samples were subsequently counterstained with hematoxylin to visualize cell nuclei, washed in running tapwater for 5 min, and mounted with Aquatex®. Imaging was performed using Nanozoomer-RS. To quantify the staining, single spheroid sections at a 20× magnification were analyzed using ImageJ software (ImageJ, NIH, USA) at a fixed threshold.
6
Histological Analysis of Osteochondral Tissue
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Frozen Sects. (10 μm) of osteochondral columns were prepared using the Kawamoto’s film method38 (link) and subjected to hematoxylin and eosin (H&E) staining, safranin O/fast green staining, and immunostaining. For the central third of the safranin O/fast green stained images, the percentage of the area stained with safranin O to the total cartilage layer was calculated using ImageJ. Histopathological evaluation of OA research society international (OARSI) osteoarthritic cartilage was performed as previously reported39 (link). For immunostaining, frozen sections were incubated in permeability buffer (1X PBS/0.2% Triton X-100) for 10 min, 0.3% H2O2 in 1 × PBS for 30 min, and Blocking One Histo (06,349–64; Nacalai Tesque, Inc., Kyoto, Japan) for 30 min at room temperature. Sections were then incubated overnight at 4 °C with the primary antibodies, such as goat anti-type I collagen (1:200; 1310–01; SouthernBiotech, Birmingham, AL, USA) and goat anti-type II collagen (1:200; 1320–01; SouthernBiotech, Birmingham, AL, USA). Immune complexes were detected using anti-goat IgG H&L (HRP) (1:400; ab97110; Abcam, Boston, MA, USA) and ImmPACT DAB (SK-4105; Vector Laboratories, Burlingame, CA, USA). Images were obtained using a DMi8 (LEICA) microscope or an Olympus BX53 microscope.
7
Immunofluorescence Staining of Extracellular Matrix
8
Antibodies for Cellular Analysis
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Antibodies used were rabbit anti‐p21 (Cell Signaling Technology, 2947), rabbit anti‐p16 (Santa Cruz Biotechnology, sc‐468), mouse anti‐β‐actin (Millipore, Billerica, MAB1501), mouse anti–α smooth muscle actin (Sigma‐Aldrich, A2547), goat anti‐type I collagen (SouthernBiotech, 1310‐01), rabbit anti‐β‐catenin (Cell Signaling Technology, 8480), rabbit anti‐non‐phospho (active) β‐catenin (Cell Signaling Technology, 8814), rabbit anti‐histone H3 (Cell Signaling Technology, 4499), mouse anti‐CD81 (Santa Cruz Biotechnology, 555675), mouse anti‐CD9 (Santa Cruz Biotechnology, sc‐59140), mouse anti‐CD63 (BD Pharmingen, 556019), rabbit anti‐Hsc70 antibody (Proteintech, 10654‐1‐AP) and mouse anti–Tom20 (Santa Cruz Biotechnology, sc‐17764). Hoechst 33242 (Sigma‐Aldrich, H342) and collagen type I solution from rat tail (Sigma‐Aldrich, C3867) were purchased reagents.
9
Whole Cell Lysate Preparation and Western Blot Analysis
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Whole cell lysates were prepared by extracting proteins using RIPA buffer containing 50 mM Tris (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 g/mL pepstatin, and 1 g/mL aprotinin. Western blot analysis was performed as previously described [13 (link)]. The protein concentration was measured with a Bradford assay (Bio-Rad, Hercules, USA), and an equal amount of each lysate was separated on a 10% SDS-polyacrylamide gel. After the proteins were transferred to polyvinylidene fluoride membranes (Millipore, MA, USA), the membranes were blocked for 1 h in TBST containing 5% skimmed milk. Blocked membranes were then incubated with primary antibodies (goat anti-type I collagen (#1310); Southern Biotechnology, Birmingham, AL, anti-phospho-ERK (#4377), anti-ERK (#9102) and anti-Ets-1 (#14069), anti-phospho-Smad2 (#3108), anti-Smad2 (#8685); Cell Signaling Technology, MA, USA,) diluted 1:1000 overnight at 4°C, washed three times with TBST for 15 min, and incubated with horseradish peroxidase-conjugated anti-goat or anti-rabbit secondary antibody (Sigma Aldrich, St. Louis, USA) diluted 1:5000 for 1 h at room temperature. The membranes were visualized using an ECL detection kit (Thermo Fisher Scientific).
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