Immunohistochemistry was performed on deparaffinized and rehydrated sections with specific primary antibodies: type II collagen antibody (anti-mouse: 1:400, ab34712, Abcam and anti-rabbit: 1:100, 08631711, MP biomedicals), aggrecan antibody (anti-mouse: 1:100, ab1031, Merck Millipore and anti-rabbit: 1:100, MA3-16888, Thermo Fisher Scientific) and NITEGE antibody (anti-mouse: 1:500, PA1-1746, Thermo Fisher Scientific and anti-rabbit: 1:50, MBS442004, My Biosource (San Diego-CA, USA) for the detection of aggrecan cleavage. All sections were counterstained using Mayer’s hematoxylin (RAL Diagnostic, Martillac, France). Tissue staining was viewed using Nanozoomer 2.0 Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). Immunostaining intensity for NITEGE was quantified by determining the relative intensity of the stained articular cartilage matrix as previously described33 (link). Briefly, images were converted into greyscale. The mean grey level values of 12 distinct regions of interest (50 × 50 pixels) from the femoral condyle and tibial plateau were determined. The value obtained was corrected by the mean of the gray levels of the extracellular matrix (10 × 10 pixels). Then, the corrected mean was subtracted from the blank (10 × 10 pixels). Finally, the value is expressed as fold increase over control condition. Measurements were performed using FIJI software.
Ma3 16888
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MA3-16888 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for use in research and scientific applications, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab34712, by Abcam (6 mentions)
Ab14106, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
Ab22552, by Abcam (2 mentions)
Sm α actin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab1031, by Merck Group (1 mentions)
Pa1 1746, by Thermo Fisher Scientific (1 mentions)
Mbs442004, by MyBioSource (1 mentions)
Nanozoomer 2.0 hamamatsu slide scanner, by Hamamatsu Photonics (1 mentions)
Pparγ, by LifeSpan BioSciences (1 mentions)
Ab58632, by Abcam (1 mentions)
Protein extraction kit, by Merck Group (1 mentions)
Hrp labeled goat anti mouse igg, by Beyotime (1 mentions)
Ab6308, by Abcam (1 mentions)
A0216, by Beyotime (1 mentions)
Hrp labeled goat anti rabbit igg, by Beyotime (1 mentions)
Sc 10758, by Santa Cruz Biotechnology (1 mentions)
Aa128, by Beyotime (1 mentions)
Mab13426, by Merck Group (1 mentions)
9 protocols using ma3 16888
1
Quantitative Evaluation of Cartilage Matrix Proteins
2
Chondrocyte Characterization by Immunofluorescence
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OA chondrocytes were identified by immunofluorescence staining of aggrecan (MA3-16888, Thermo Fisher Scientific, USA), collagen II (Origene, USA), and PPARγ (LifeSpan, USA).20 (link)
Chondrocytes were fixed with 4% paraformaldehyde for ten minutes, and then blocked with 1% bovine serum (BSA) for one hour at room temperature. The chondrocytes were labelled for two hours at 37°C with antibodies specific for aggrecan, collagen II, and PPARγ at 2 µg/ml in 0.1% BSA. Chondrocytes were then incubated with FITC-conjugated secondary antibodies (Agrisera, Sweden; 1:200) for one hour at 37°C. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for five minutes in the absence of light. Following nuclear DAPI counterstaining, the number of positive cells among the total were counted in ten randomly selected fields per culture dish.
Chondrocytes were fixed with 4% paraformaldehyde for ten minutes, and then blocked with 1% bovine serum (BSA) for one hour at room temperature. The chondrocytes were labelled for two hours at 37°C with antibodies specific for aggrecan, collagen II, and PPARγ at 2 µg/ml in 0.1% BSA. Chondrocytes were then incubated with FITC-conjugated secondary antibodies (Agrisera, Sweden; 1:200) for one hour at 37°C. Nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) for five minutes in the absence of light. Following nuclear DAPI counterstaining, the number of positive cells among the total were counted in ten randomly selected fields per culture dish.
3
Quantitative Protein Expression Analysis
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The total protein was collected using a protein extraction kit (Sigma-Aldrich, USA). The protein concentrations were determined using the BCA assay (Pierce, Thermo Fisher, USA). After denaturing at 95 °C for 5 mins, 5 mg aliquot of total protein of each sample was subjected to the 4–20% gradient SDS-PAGE, transferred to a PVDF membrane (Millipore, USA). The membrane was incubated in blocking solution with donkey serum (GTX30972, GeneTex, USA) for 1 hour, and was incubated overnight at 4 °C with the primary antibodies against Col X (ab58632, Abcam, USA), Col I (ab6308, Abcam, USA), Col II (ab116242, Abcam, USA), Sox 9 (PA5-23383, ThermoFisher, USA), Aggrecan (MA3-16888, ThermoFisher, USA), Notch1-ICD (07–1232, Millipore, USA), MMP-13 (MAB13426, Millipore, CA), Runx2 (sc-10758, Santa Cruz, USA) and β-actin (AA128, Beyotime, China). One of the following secondary antibodies was used: HRP-labeled goat anti-rabbit IgG (A0208, Beyotime, China) and HRP-labeled goat anti-mouse IgG (A0216, Beyotime, China). Proteins in the blots were visualized by an ECL plus kit (P0018, Beyotime, China). The integrated optical density of bands was quantified with the ImageJ software. Each sample was normalized to β-actin.
4
Osteoarthritis Therapeutic Evaluation Protocol
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Pam3CSK4 (Pam3; tlrl-pms) was purchased from Invitrogen. DL-5-MTP (28052-84-8) was purchased from Sigma-Aldrich. Pure L form of 5-MTP (L-5-MTP) and 5-MTP methylester analog, L-5-MTPE were commercially synthesized by ASTATECH, USA. Antibodies for OPG (TA322994; Origene), α-SM actin (A5228; Sigma-Aldrich), SM22α (ab14106; Abcam), osterix (ab22552; Abcam), collagen II (ab34712; Abcam), SOX9 (sc-20095; Santa Cluz), aggrecan (MA3-16888; Thermo), interleukine-6 (ab6672; Abcam), pp38 (#4511; Cell Signaling Technology), p38 (sc-7194; Santa Cluz), pCREB (#9198; Cell Signaling Technology), CREB (#9197; Cell Signaling Technology), pp65 (#3033; Cell Signaling Technology), p65(#4746; Cell Signaling Technology) and β-actin (MAB1501; Millipore) were used in Western blot analysis and Immunohistochemistry. The antibody for detecting 5-MTP was customized by GeneScript, USA. Powders of Alizarin red S (ARS; A5533) and Alcian blue (A5268) were purchased from Sigma-Aldrich.
5
Quantifying Collagen II and Aggrecan
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The Col II and Agg production was evaluated by WB at 21 days of co-culture. Briefly, protein extracts were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Immobilon P, USA). The membranes were incubated with anti-Collagen II (ab34712, Abcam, UK) and anti-Aggrecan (MA3-16888, Thermo Fisher, USA) at 4 °C overnight and with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA) at 37 °C for 1 h. Protein bands were imaged by ChemiDoc XRS Plus luminescent image analyzers (Bio-Rad, Hercules, CA, USA). The protein levels were normalized to GAPDH.
6
Signaling Pathway Activation Analysis
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Pam2CSK4 (Pam2), Pam3CSK4 (Pam3), zymosan, and HSP60 (heat shock protein 60) were purchased from Invitrogen. U0126 was from InvivoGen. SB203580, PD98059, SP600125, and LY294002 were from Calbiochem. Antibodies for OPG (TA322994; Origene) Runx2 (TA309842; Origene), α-SM actin (A5228; Sigma-Aldrich), SM22α (ab14106; Abcam), osterix (ab22552; Abcam), Collagen II (ab34712; Abcam), SOX9 (sc-20095; Santa Cluz), aggrecan (MA3-16888; Thermo), and β-actin (MAB1501; Millipore) were used in Western blot analysis and Immunohistochemistry.
7
Immunofluorescence Staining of Condylar Cartilage
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Cell, rat and human condylar samples were fixed with 4% paraformaldehyde for 48 h. Rat and human condyle pieces were decalcified in 15% ethylenediaminetetraacetic acid (EDTA) at 4 C for 8e10 weeks. Samples were embedded in paraffin and 5 mm sections were cut. Sections were incubated in blocking buffer (5% goat serum) for 1 h at room temperature, before incubation with rabbit anti-GFP (1:200, Abcam), rabbit anti-SOX9 (1:100, ab3697, Abcam) and mouse anti-Aggrecan (1:200, MA3-16888, Invitrogen) overnight at 4 C. Sections were then stained at room temperature for 2 h with Alexa Fluor 568 goat anti rabbit IgG antibody (1:500, A-11036, Invitrogen), Alexa Fluor 568 goat anti mouse IgG antibody (1:500, A-11004, Invitrogen), Alexa Fluor 488 goat anti mouse IgG antibody (1:500, A32723, Invitrogen) and Alexa Fluor 488 goat anti rabbit IgG antibody (1:500, A-11008, Invitrogen). DAPI (H-1200, Vector Laboratories) was used for nuclear counterstaining.
8
Characterization of Engineered Cartilage Tissue
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At the end of the chondrogenesis or growth culture, the final tissue constructs were washed with PBS and fixed with 95% v/v ice ethanol at 4 °C for 20 min. Afterwards, samples were permeabilized with 0.3% v/v Triton X-100 at room temperature for 20 min and washed three times (5 min each time) with PBS. Then the samples were blocked in 3% bovine serum albumin (BSA, #ST023, Beyotime, China) solution for 1.5 h at room temperature. The tissue constructs were then incubated with primary antibody for collagen II (1:800 v/v, mouse collagen II monoclonal antibody, #ab34712, Abcam, UK) and ACAN (1:400 v/v, rabbit ACAN monoclonal antibody, #MA3-16888, Invitrogen, USA) for 2 h at room temperature. The samples were washed three times with 0.2% v/v Tween-20 in PBS and incubated with goat anti-mouse IgG secondary antibody (1:500 v/v, #ab150115, Abcam, UK) and goat anti-rabbit IgG secondary antibody (1:500 v/v, #ab150080, Abcam, UK) in the dark for 2 h at room temperature. Hoechst fluorescent dye (1.5 µg/mL in PBS, #B1123845, Aladdin, China) was used to counterstain nuclei. The samples were then washed five times with 0.2% v/v Tween-20 in PBS, and imaged using laser confocal microscope (Olympus FV3000, Japan).
9
Western Blot Analysis of Cartilage Proteins
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Samples were lysed in ice RIPA lysis buffer (#P0013B, Beyotime, China), incubated on ice for 30 min, and centrifuged at 12,000g for 10 min. The protein concentration was determined with a bicinchoninic acid (BCA) protein assay (#P0010, Beyotime, China). Equal amounts of protein extracts were fractionated by 12% sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with the following primary antibodies overnight at 4 °C: collagen II (mouse collagen II monoclonal antibody, #ab34712, Abcam, UK) and aggrecan (rabbit ACAN monoclonal antibody, #MA3-16888, Invitrogen, USA), ERK (rabbit monoclonal ERK antibody, #4695, Cell Signaling Technology, USA) and pERK (rabbit monoclonal pERK antibody, #9101, Cell Signaling Technology, USA). GAPDH was used as an internal loading control and detected with a mouse monoclonal antibody (#ab8245, Abcam, UK). The secondary HRP-conjugated antibody (goat anti-rabbit antibody, #BK-R050; goat anti-mouse #BK-M050, Bioker, China) was used for detection. The antibody associated protein bands were revealed using the Ncm ECL Ultra Western blotting kit (#P10100, Ncm Biotech, China), and visualized using the Touch Imager XLi (e-BLOT, China).
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