For IHC analysis of Wnt7a and GFP proteins, heat-induced antigen retrieval in 10 mM citric acid buffer was performed. For Collagen II staining, enzymatic antigen retrieval was done using 0.3% hyaluronidase and 0.15% trypsin (Invitrogen) at 37 °C. Sections were incubated with the following primary antibodies overnight at 4 °C: Wnt7a (Santa Cruz), GFP (Abcam), Collagen II (Abcam); and the following secondary antibodies for 2hrs: Wnt7a staining–donkey anti-goat IgG (H + L) secondary antibody Texas Red conjugate (Jackson) or a biotin-conjugated secondary antibody (Vector) for chromogenic staining; Collagen II staining - goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor® 594 conjugate (Invitrogen); GFP - goat anti-chicken IgY (H + L) secondary antibody, Alexa Fluor® 488 conjugate (Invitrogen). Fluorescent sections were counterstained with DAPI. The Vectastain Elite ABC Kit (Vector) was used for chromogenic staining. Sections incubated without the primary antibody served as negative controls for each round of staining.
Collagen 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Collagen II is a type of collagen protein found in the extracellular matrix of cartilage, bone, and other connective tissues. It is a key structural component that provides strength and resilience to these tissues. This lab equipment product can be used for various research applications that involve the study and analysis of collagen II.
Automatically generated - may contain errors
Lab products found in correlation
Biotin conjugated secondary antibody, by Vector Laboratories (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Collagen 2, by Abcam (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Wnt7a, by Santa Cruz Biotechnology (1 mentions)
Bx61 microscope, by Olympus (1 mentions)
Dp71 digital camera, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
Twist1, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase labelled secondary antibody, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Aggrecan, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
C ebpβ, by Cell Signaling Technology (1 mentions)
Mmp 3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
5 protocols using collagen 2
1
Immunohistochemical Staining of Wnt7a, GFP, and Collagen II
2
Immunofluorescence Staining of Cell Cultures
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Cell immunofluorescence was performed as previously described [7 (link)]. Fixed and membrane-permeabilized cells were incubated with primary antibodies for 1 h at RT, and with fluorescent-secondary antibodies for another 1 h at RT, in dark. Nuclei were stained with 4’,6-diamidino-2- phenylindole dihydrochloride (DAPI; Sigma-Aldrich) for 4 min at RT in the dark. The following primary antibodies were used: Cx43 (Sigma-Aldrich, C6129; 1:500), collagen II (Invitrogen, Thermo Fischer Scientific, MA5-12789; 1:200), NF-κB (Santa Cruz Biotechnology, sc-8008; 1:100) and Twist-1 (Santa Cruz Biotechnology, sc-81417; 1:100). Goat anti-rabbit FITC-conjugated (F-2765; 1:100) and goat anti-mouse Alexa 594-conjugated (A-11032; 1:200) secondary antibodies were used (both from Invitrogen, Thermo Fisher Scientific). Negative controls without primary antibody were performed to test the specificity of each antibody. The samples were analysed on an Olympus BX61 microscope using a DP71 digital camera (Olympus). A minimum of n = 3 experiment were performed.
3
Xanthohumol Modulates Inflammatory Responses in Chondrocytes
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Xanthohumol (XH) (purity98%) (Cat. no. D108095) was obtained from BIOBW (Beijing, China). Collagenase type II (Cat. no. C2-28) and dimethylsulfoxide (DMSO) (Cat. no. D8418) were purchased from Sigma Aldrich (St Louis, MO, United States). The primary antibodies against C/EBPβ (Cat. no. 3082), HO-1 (Cat. no. 43966), MMP-3 (Cat. no. 14351), GAPDH (Cat. no. 5174S), and the horseradish peroxidase-labelled secondary antibody (Cat. no. 7074S) were purchased from Cell Signaling Technology. MMP-13 (Cat. no. 701287), ADAMTS-4 (Cat. no. PA5-69140), ADAMTS-5 (Cat. no. PA5-32142), collagen-II (Cat. no. MA5-12789), and aggrecan (Cat. no. MA3-16888) were obtained from Invitrogen. Recombinant rat IL-1β (Cat. no. ab9788) purchased from Abcam.
4
Protein Expression Analysis in ATDC5 Cells
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Total proteins in whole-cell lysates of ATDC5 cells were extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime). After centrifugation at 400 × g at 4°C for 15 min, the protein concentration of the extracts was determined using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime). A sample of 60 μg total protein was subjected to 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% fat-free milk for 1 h and then incubated with primary antibodies against TBK1 (1: 500, BosterBio), ADAMTS-4 (1:500, Abcam), MMP3 (1:1,000, Abcam), MMP13 (1:1,000, Abcam), SOX9 (1:1,000, Abcam), aggrecan (1:1,000, Abcam), p-JAK1 (phospho Y1022 + Y1023) (1:1,000, Abcam), JAK1 (1:1,000, Abcam), p-JAK2 (phospho Y1007 + Y1008) (1:1,000, Abcam), JAK2 (1:1,000, Abcam), p-STAT3 (phospho Y705) (1:1,000, Abcam), STAT3 (1:1,000, Abcam), collagen II (1:1,000, Thermo Fisher Scientific) and GAPDH (1:2,000, Thermo Fisher Scientific) at 4°C overnight. After incubation with the secondary antibodies for 1 h, the blots were visualized using a chemiluminescent agent (ECL, Millipore, USA).
5
Protein Expression Analysis of CHON-001 Cells
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CHON-001 cells were lysed with RIPA buffer (Beyotime) and total protein was collected. Approximately 20 μg of protein were separated using 10% SDS-PAGE and then electro-transferred to PVDF membranes (Roche, Basel, Switzerland). After blocking with blocking buffer for western blotting (Thermo Fisher Scientific), membranes were incubated with primary antibodies against MMP-13 (1:2000, PA5-27242, Thermo Fisher Scientific), ADAMTS-5 (1:1000, PA5-14350, Thermo Fisher Scientific), Collagen II (1:2000, MA5-12789, Thermo Fisher Scientific), NLRP3 (1:1500, MA5-23919, Thermo Fisher Scientific), cleaved-caspase-1 (1:1000, PA5-105049, Thermo Fisher Scientific), and β-actin (1:4000, ab8226, Abcam, CA, USA) overnight at 4 °C. After washing thrice with PBST, membranes were incubated with HRP-labelled anti-mice/rabbit IgG secondary antibody. The protein blots were visualized with an enhanced chemiluminescence reagent (Pierce, IL, USA).
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