The expression levels of SUMO2/3, aggrecan, and collagen type II in primary human chondrocytes were analyzed by immunofluorescence microscopy. After washing three times with PBS, cells were fixed with 4% formaldehyde for 10 mins at room temperature, followed by blocking with 2.5% BSA for 1 hr at room temperature. Cells were subsequently incubated with anti-SUMO 2/3 (1:500; ab3742; Abcam), anti-aggrecan antibody (1:500; ab3778; Abcam), and anti-collagen type II antibody (1:500; ab3778; Abcam) overnight at 4°C in DPBS containing 2.5% BSA and 0.1% Tween 20. Samples were then washed three times with Dulbecco's Phosphate-Buffered Saline (DPBS), followed by probing with Fluorescein isothiocyanate (FITC) or Tetramethylrhodamine (TRITC) conjugate secondary antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 hr. Thereafter, cells were counterstained with DAPI and analyzed using a fluorescence microscope.
Ab3778
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab3778 is a rabbit monoclonal antibody targeted against the human CD3 epsilon protein. It is intended for use in various immunoassay applications, including flow cytometry, immunohistochemistry, and Western blotting. The antibody recognizes a specific epitope on the CD3 epsilon chain, which is a component of the T-cell receptor complex.
Automatically generated - may contain errors
Lab products found in correlation
Ab34712, by Abcam (27 mentions)
Ab41037, by Abcam (16 mentions)
Ab188570, by Abcam (14 mentions)
Ab39012, by Abcam (14 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Ab8245, by Abcam (7 mentions)
Ab185230, by Abcam (5 mentions)
Ab51072, by Abcam (4 mentions)
Ab219620, by Abcam (4 mentions)
Ab185722, by Abcam (3 mentions)
Ab2302, by Abcam (3 mentions)
Aggrecan, by Abcam (3 mentions)
Ab52915, by Abcam (3 mentions)
Ab185430, by Abcam (3 mentions)
Ab6721, by Abcam (3 mentions)
Ab16663, by Abcam (3 mentions)
Ab150113, by Abcam (2 mentions)
Ab150077, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
63 protocols using ab3778
1
Quantifying Chondrocyte Extracellular Matrix
2
Protein Extraction and Western Blot Analysis
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The cells were lysed for 30 min using RIPA reagent (Beyotime, China) containing protease inhibitors (Beyotime) on ice, and then the supernatant was collected by centrifugation (12,000 rpm, 5 min, 4°C). The protein concentration in the supernatant was detected by BCA detection kit (Beyotime, China). 25 μg protein per well was subjected to SDS-PAGE and then electrically transferred to PVDF membranes (Millipore, USA). The membranes were sealed with 5% skimmed milk powder for 1 h, the corresponding primary antibody [cleaved caspase 3 (1:1000, ab2302, Abcam), cleaved caspase 9 (1:1000, ab2324, Abcam), aggrecan (1:1000, ab3778, Abcam), collagen II (1:1000, ab34712, Abcam), MMP13 (1:1000, ab51072, Abcam), ADAMTS4 (1:1000, ab185722, Abcam), LC3A/LC3B (1:1000, PA1-16,930, Invitrogen), p62 (1:1000, PA5-20,839, Invitrogen), GAPDH (1:2000, ab8245, Abcam)] was added and incubated overnight at 4°C, and the secondary antibody [goat anti-rabbit (1:2000, ab150077, Abcam), goat anti-mouse (1:2000, ab150113, Abcam)] was added and incubated at room temperature for 2 h, the protein bands were developed with ECL reagent (Beyotime), and the gray value was quantified using image J 6.0 (NIH).
3
Protein Expression Analysis in Cartilage
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Total protein was extracted using RIPA lysis buffer (C0481, Sigma-Aldrich) replenishing 1% complete protease and phosphatase inhibitor (Beyotime). The concentration of each protein sample was determined using the bicinchoninic acid (BCA) kit (23227, TH&Ermo). The protein was loaded on the SDS-PAGE gel and transferred to the PVDF membrane (Millipore). The membrane was blocked with 5% BSA at ambient temperature for 1 h and incubated with the primary antibodies (all from Abcam) overnight at 4°C and horseradish peroxidase-labeled goat anti-rabbit IgG (ab205718, 1 : 2000, Abcam) or goat anti-mouse IgG (ab197767, 1 : 2000, Abcam) for 1.5 h at room temperature. Afterwards, the bands were visualized using chemiluminescent solution (NCI4106, Pierce, Rockford, IL). ImageJ 1.48 software (National Institutes of Health, Bethesda, Maryland) was adopted for band analysis, and protein expression was normalized to GAPDH expression. The primary antibodies (all from Abcam) were rabbit anti-MMP13 (ab84594, 1 : 1000), rabbit anti-ADAMTS 5 (ab41037, 1 : 500), rabbit anti-COL II (ab188570, 1 : 1000), rabbit anti-aggrecan (ab3778, 1 : 1000), and rabbit anti-GAPDH (ab9485, 1 : 2500).
4
Immunofluorescent Localization of ECM Proteins
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4-µm wax sections were deparaffinized, hydrated, and preincubated with 10% goat serum in PBS for 1 h at RT. Sections were incubated overnight at 4°C with primary antibodies (FGF-2, Abcam; ab8880 at 1:100 dilution; perlecan, Invitrogen; MA5-14641 at 1:100 dilution; and aggrecan, Abcam; ab3778 at 1:150) in 10% goat serum followed by a wash and incubation with their corresponding secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 (Life Technologies; 1;100 dilution) for 1 h at RT. Images were captured with a Zeiss LSM700 confocal microscope using Zen blue software. A 20×/0.8-NA objective was used, with air medium at RT.
5
Histological Analysis of Redifferentiated Chondrocytes
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At day 14 of redifferentiation, the alginate beads of all three donors and six conditions were fixed overnight in 100 mM CaCl2 in 4% neutral buffered formaldehyde solution and thereafter dehydrated in ethanol.43 The next day, the alginate beads were embedded in paraffin, 5 μm sections were cut and stained with H&E (109249 and 115935, Merck). Immunohistochemistry (IHC) staining for collagen type I and II was performed as described previously.44 IHC for aggrecan (mouse monoclonal recombinant antibody, 4 μg/ml; ab3778, Abcam) was performed for collagen type I and II, but with 60 instead of 30 min of 1 mg/ml pronase and 10 mg/ml hyaluronidase antigen retrieval treatment. Cytokeratin 19 (mouse monoclonal antibody, 10 μg/ml; ab9221, Abcam) IHC was performed without any antigen retrieval. Cytokeratin 8 (mouse monoclonal antibody, 10 μg/ml; MA1‐19037, Thermofisher) and PAX1 (rabbit polyclonal, 4 μg/ml; ab203065, Abcam) IHC was performed with 10 mM citrate buffer (pH 6, 70°C) antigen retrieval for 60 and 30 min, respectively. As negative control in all IHCs, normal mouse IgG1 (X0931, Dako), or Rabbit IgG polyclonal isotype control (ab37415, Abcam) was applied at a similar concentration as the primary antibody.
6
Western Blot Analysis of Cartilage Proteins
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Cartilage tissues and primary chondrocytes were lysed to obtain protein extracts, which were subjected to SDS‐PAGE (8%‐12% gel) and then transferred to polyvinylidene fluoride membranes (Millipore: Billerica, MA, USA). Membranes were blocked for 1 hour with 5% non‐fat milk prepared in Tris‐buffered saline (TBS) containing 0.1% Tween 20 (TBST) at room temperature (RT). Next, membranes were incubated overnight at 4°C with specific primary antibodies against PIK3R1 (Proteintech: Chicago, IL, USA, 60225‐1‐I, 1:500), cleaved caspase‐3 (Cell Signaling: Beverly, MA, USA, #9661, 1:1000), collagen Ⅱ (Novus Biologicals: Littleton, CO, USA, NBP1‐77795, 1:2000), aggrecan (abcam, ab3778, 1:1000), MMP3 (abcam: Cambridge, MA, USA, ab52915, 1:2000), MMP13 (abcam, ab39012, 1:2000) and GAPDH (Santa Cruz Biotechnology: Santa Cruz, CA, USA, sc‐32233, 1:5000). After washing with TBST, membranes were incubated with horseradish peroxidase‐labelled secondary antibodies at RT for 1 hour. The signal was visualized using an enhanced chemiluminescence reagent according to the manufacturer's protocol (Millipore: Billerica, MA, USA). GAPDH served as a loading control. The protein expression was quantified by ImageJ (http://rsb.info.nih.gov/ij/ ).
7
Western Blot Analysis of Cartilage Proteins
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For the validation of the differentially expressed proteins, western blotting was used for quantitative analysis. Cartilage tissues were lysed by 2% sodium dodecyl sulfate with 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride, then the lysates were centrifuged. The supernatants were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skimmed milk, the membrane was then analyzed using specific antibodies and visualized by enhanced chemiluminescence (ECL Kit, Amersham Biosciences, Little Chalfont, UK). The primary antibodies to Indian hedgehog (IHH, ab52919), type I collagen (COL1A1, ab138492), type II collagen (COL2A1, ab188570), aggrecan (ACAN, ab3778) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab9894) were obtained from Abcam (Cambridge, UK). Protein signals were detected using secondary HRP-conjugated anti-mouse or anti-rabbit antibodies by the enhanced chemiluminescence (ECL Kit, Amersham Biosciences, Little Chalfont, UK). The full-length images of individual blots were displayed in Fig. S1 .
8
Histological Evaluation of Rat Joint Tissues
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The joint samples, heart, liver, spleen, lung, kidney and skin tissue were collected from the rats and routine HE staining was performed. Knee joint samples were fixed in 4% (v/v) neutral buffered formalin for 24 h and decalcified for 1 month at room temperature in neutral 10% EDTA solution. Subsequently, the sample was dehydrated in an ethanol gradient, clarified, and embedded in a paraffin block. Tissue sections (8 μm) were prepared. Six representative sections of the joints from various depths were mounted on glass slides, stained with Safranin-O, and photographed under a microscope (BH2 UMA, Olympus). After overnight incubation with MMP13 (ABCAM, ab39012), COL2A1 (ABCAM, ab34712), ADAMTS-5 (ABCAM, ab41037), and AGGRECAN (ABCAM, ab3778) primary antibodies at 4°C, sections were incubated with secondary antibodies for 2 h at room temperature. Color development was performed using a DAB substrate system. Hematoxylin was used to stain the nucleus of cells.
9
Quantitative Protein Analysis of Cartilage
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Cellular protein from cells or tissue prepared using buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) was separated by SDS-PAGE following standard protocols (Amersham BioSciences, Piscataway, NJ, USA). The antibodies used for Western blotting were as follows: aggrecan (ab3778; Abcam, Cambridge, UK), collagen type II (ab3778; Abcam), MMP-13 (sc-515,284; Santa Cruz Biotechnology, Dallas, TX, USA), ADAMTS-4 (MAB4307; R&D Systems, Minneapolis, MN, USA), ADAMTS-5 (MAB2198; R&D Systems), and GAPDH (sc-166,574; Santa Cruz). An enhanced chemiluminescence system (Amersham BioSciences) was used for detection. Blots were quantified using the ImageJ densitometry program (National Institutes of Health, Bethesda, MD, USA).
10
Western Blot Analysis of Cartilage Proteins
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The proteins were isolated by RIPA buffer (Beyotime). The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). These membranes were then blocked with 5% non-fat milk (Beyotime) and incubated with primary antibodies overnight at 4℃.The primary antibodies, including anti-SOX9 (1:1000, ab185966, Abcam), anti-PCNA (1:1000, 13110S, CST), anti-cleaved caspase-3 (1:1000, 9661S, CST), anti-MMP13 (1:1000, 69926S, CST), anti-ADAMTS5 (1:1000, ab41037, Abcam), anti-COL2A1 (1:1000, ab188570, Abcam), anti-Aggrecan (1:1000, ab3778, Abcam), or anti-β-actin (1:2,000, ab8227, Abcam). After secondary antibodies were incubated with the membranes, protein bands were developed with an ECL kit (Solarbio, Beijing, China).
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