Indirect immunoperoxidase labeling was carried out using human specific antibodies against collagens type I (COL-1, Sigma Aldrich, UK), II (Abcam, UK), X (Gift from Klaus von der Mark), and aggrecan (5C5, Enzo Life Sciences, UK). Fluorescence labeling was used to detect Stro-1 (R&D Systems, UK). Primary antibodies were diluted in 0.1 M PBS containing 0.01% Tween 20 (PBS-T) at a concentration of 10 µg mL−1. Appropriate antigen retrieval techniques were necessary to expose the collagen, aggrecan, and Stro-1 epitopes. For collagen types I and II, pellets were subjected to a chondroitinase (0.25 U mL−1; Sigma Aldrich, UK) and hyaluronidase (2 U mL−1; Sigma Aldrich, UK) pretreatment for 1.5 hours at 37°C. For collagen type X, pellets were first subjected to a proteinase K (2.0 µg mL−1; Sigma Aldrich, UK) digest for 1 hour at 37°C, and subsequently a chondroitinase (0.25 U mL−1) and hyaluronidase (2 U mL−1) treatment for 30 minutes at 37°C. For aggrecan (5C5), pellets were subjected to a hyaluronidase (2 U mL−1) pretreatment for 1.5 hours at 37°C. Pellets from a minimum of three different clonal cell lines were labeled for each antibody. For Stro-1, pellets were pretreated with 0.3% triton X (in PBS) for 30 minutes at room temperature.
Chondroitinase
Manufactured by Merck Group
Sourced in United Kingdom, United States
Chondroitinase is a laboratory enzyme used to digest chondroitin sulfate, a major component of the extracellular matrix in various tissues. It is commonly used in research applications to study the role of chondroitin sulfate in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (3 mentions)
Lsm 780, by Zeiss (2 mentions)
Luna c18, by Phenomenex (1 mentions)
Powerlab system, by ADInstruments (1 mentions)
Infinite 200 microplate reader, by Tecan (1 mentions)
I control software, by Tecan (1 mentions)
Fluorescently labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cryotome, by Leica (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Dfc 3200 camera, by Leica (1 mentions)
Ckx41 microscope, by Leica (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ii ii6b3, by Developmental Studies Hybridoma Bank (1 mentions)
H2519, by Merck Group (1 mentions)
Hoechst dye, by Merck Group (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Ab5535, by Merck Group (1 mentions)
Ab1031, by Merck Group (1 mentions)
α sma, by Abcam (1 mentions)
10 protocols using chondroitinase
1
Immunohistochemical Characterization of Chondrogenic Cells
2
Quantifying Chondroitin-6-Sulfate in Silk Fibroin
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Chondroitinase (Sigma, Taufkirchen, Germany), Waters’ 515 pump, controller, and 486 UV detector were used for HPLC analysis to measure chondroitin-6-sulfate content. The fibroin bioadhesive was prepared and inserted into the columns for analysis using a Phenomenex Luna C18. Silk fibroin protein extracts were analyzed using HPLC, and a graph comparing the concentrations of chondroitin-6-sulfate in silk fibrin (negative control), silk fibroin protein, and silk fibroin protein with hydroxyapatite was plotted. MeOH:KH2PO4 = 1:4 v/v was used as the mobile phase, and 0.1, 0.2, 0.4, and 0.8 wt% of samples and the standards were dissolved in HPLC water for analysis. The area was calculated based on the concentration.
3
Enzymatic Depletion of Endothelial Glycocalyx
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Seven-week-old male Sprague Dawley rats (approximately 250 g; Harlan, Bicester, UK) were euthanised by cervical dislocation and hearts quickly removed, mounted on a Langendorff apparatus and perfused in a non-recirculating mode with Krebs solution as described previously [30 (link)]. Contractile function was measured using a latex balloon in the left ventricle. Data acquisition and analysis used a PowerLab System (AD Instruments, Bella Vista, NSW, Australia). Measurement was initiated after hearts were stabilised by perfusion for 30 min. Hearts were then perfused for 40 min with the combination of hyaluronidase (14 μg/ml; Sigma Aldrich, UK) and chondroitinase (0.0022 u/ml; Sigma Aldrich) in Krebs solution to deplete eGlx or with Krebs solution alone as control. We have shown previously that this combination of enzymes reduces eGlx thickness and coverage in glomerular capillaries [21 (link)]. Some isolated hearts were also perfusion-fixed with glutaraldehyde and Alcian Blue for analysing eGlx by electron microscopy as above.
4
Chondroitin Sulfate Assay in Urine
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DMMB zinc chloride double salt and Chondroitin sulphate A (CS) sodium salt from bovine trachea were purchased (Sigma-Aldrich, St. Louis, MO, USA). The DMMB reagent was prepared according to a standard protocol, in which DMMB (16 mg), glycine (3.05 g), sodium chloride (1.6 g) and acetic acid (544 μL) were made up to a volume of 1 L with distilled water and stored at 4 °C without exposure to light for up to a month or until precipitation occurred [11 (link),12 (link)].
For the assay, 10 µL of the dilution series or urine samples was transferred to a 96-well microplate for duplicate measurements. DMMB solution was brought to room temperature, and then 200 µL was added using an Eppendorf multidispenser. The absorbance was read immediately using a Tecan infinite 200 microplate reader and the i-control™ software (Tecan, 2.0.10.0, Männedorf, Switzerland). For each experiment, a dilution series of CS was prepared to establish a standard curve from 0 to 125 µg/mL.
In further experiments, urine samples were spiked with CS (Sigma-Aldrich, St. Louis, MO, USA), albumin (5 mg/mL), glucose (10 mg/mL), calcium chloride (3 mg/mL) or acetone (7.9 mg/mL). Chondroitinase (Sigma-Aldrich, St. Louis, MO, USA) and deoxyribonuclease I were used for enzymatic digestion of CS or DNA, respectively, in urine samples.
For the assay, 10 µL of the dilution series or urine samples was transferred to a 96-well microplate for duplicate measurements. DMMB solution was brought to room temperature, and then 200 µL was added using an Eppendorf multidispenser. The absorbance was read immediately using a Tecan infinite 200 microplate reader and the i-control™ software (Tecan, 2.0.10.0, Männedorf, Switzerland). For each experiment, a dilution series of CS was prepared to establish a standard curve from 0 to 125 µg/mL.
In further experiments, urine samples were spiked with CS (Sigma-Aldrich, St. Louis, MO, USA), albumin (5 mg/mL), glucose (10 mg/mL), calcium chloride (3 mg/mL) or acetone (7.9 mg/mL). Chondroitinase (Sigma-Aldrich, St. Louis, MO, USA) and deoxyribonuclease I were used for enzymatic digestion of CS or DNA, respectively, in urine samples.
5
Histological Analysis of Cartilage Tissue
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Following fixation, the samples were washed with PBS (Life Technologies), decalcified for 4 days in Versenate EDTA solution (American Master Tech). Processed samples were then taken through a sucrose gradient (10, 20, 30%), embedded in OCT compound (Tissue-Tek), sectioned (16 µm thick) on a cryotome (Leica), and mounted on glass slides (n = 4 section per sample). Antigen retrieval was performed with 1 mg/ml chondroitinase (Sigma-Aldrich) and 5 mg/ml hyaluronidase (Sigma-Aldrich) for 30 min at 37°C. Nonspecific binding was suppressed with 1% horse serum (Vector Labs) in PBS for 45 min. Slides were then washed with 0.1% Triton X-100/TBS, blocked in 1% BSA, incubated with primary antibodies against collagen type II (Col2) (Abcam), myosin heavy chain (MHC) (Developmental Studies Hybridoma Bank), and/or proliferating cell nuclear antigen (PCNA) (Abcam) overnight at 4°C, and incubated with fluorescently labeled secondary antibodies (Invitrogen) for 1 h at room temperature. Samples were counterstained with DAPI (Invitrogen) and imaged with an Olympus CKX41 microscope outfitted with a Leica DFC 3200 camera.
6
Histological Analysis of Cartilage Constructs
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Pellets and scaffold-based constructs were removed from media, fixed in 10% wt/vol buffered formalin (3.8% wt/vol formaldehyde), processed into paraffin wax, and sectioned at a thickness of 5 μm. Sections were stained with 0.1% wt/vol safranin O to reveal proteoglycan matrix depositions and counterstained with 1% wt/vol fast green. Other sections from scaffold-based constructs were treated with chondroitinase (Sigma-Aldrich) at 0.2 U per section and incubated with an antibody against collagen II (II-II6B3 from Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA, USA; 1:50 dilution). Immune-localized antigens were visualized with horse anti-mouse IgG biotinylated secondary antibody (Vectastain; Vector Laboratories Inc., Burlingame, CA, USA) and an aminoethylcarbazole (AEC)-based peroxidase labeling kit (Enzo Life Sciences Inc., Farmingdale, NY, USA). Images were captured by using an Eclipse Ti-S microscope (Nikon Canada Inc., Mississauga, ON, Canada) fitted with NIS Elements Basic Research Imaging Software Version 4.20 (Nikon Canada Inc.) and assembled in Photoshop.
7
Heparinase I and Chondroitinase Modulate PCSK9 Binding
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HepG2 cells stably transfected with PCSK9 were seeded at 50,000 cells per coverslip and incubated overnight before addition of Heparinase I (Sigma Aldrich/H2519) or Chondroitinase (Sigma Aldrich/C3667) in PBS (0.0002 U/ml) or PBS alone. Cells were incubated for 1 h at 37 °C, before fixation in 4% paraformaldehyde and immunostaining of non-permeabilized cells with primary and secondary antibodies. Nuclei were visualized with Hoechst dye (Sigma Aldrich). Images were acquired on a Zeiss LSM780. In a different experiment, HepG2 cells were incubated with Heparinase I before washing with 3× PBS and incubation with PCSK9 (500 nM) on ice. Following binding of PCSK9 cells were fixed and non-permeabilized cells were subjected to immunofluorescence staining.
8
Chondroitinase Injection and Imaging of Zebrafish Otocyst
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The dechorionated embryos at 48 hpf were soaked in 1X tricaine and immobilized in a 1.5% canyon mount filled with egg water. Chondroitinase (Chase from Sigma Aldrich) was dissolved in a 0.01% BSA solution and stored at 50 units/mL at −20°C. The injection solution was made with a buffer containing 50 mM Tris-Hcl, 60 mM sodium acetate, 0.02% BSA, and 0.5% Phenol Red. Chase (10 units/mL) was injected into the periotic space (anterior and posterior to the OV) of embryos at 48 hpf. The embryos were then fixed with 4%PFA/PBS at 3 hours post-injection, followed by immunostaining. To measure bud length and bud-luminal volume, 3 kDa Texas-red Dextran (Thermo Fisher, 500 μM) was injected into the periotic space of embryos at 48 hpf, followed by confocal imaging of OVs. Chase (10 units/mL) was then injected with 3kDa of Texas-red Dextran into periotic space, and OVs were captured again by confocal imaging at 3 hours post-injection.
9
Histological Analysis of Cardiac Remodeling
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ScxCre;CaV1.2TS/+ and ScxCre;CaV1.2WT/+ mice and their control littermates (CaV1.2TS/+ and CaV1.2WT/+) were sacrificed. Hearts were removed and fixed in 4% paraformaldehyde for 1 hour at room temperature. Tissue was further dehydrated through ethanol gradient washes, embedded in paraffin, and sectioned at 10 μm. Movat’s pentachrome staining was performed according to the manufacturer’s protocol (American MasterTech Scientific). For Aggrecan (1:500; Millipore, AB1031) and Sox9 (1:500; Millipore, AB5535) staining, tissues were treated with chondroitinase (200 mU/mL; Sigma) before blocking. For Runx2 (1:100; Novus Biologicals, NBP1-77472) and α-SMA (1:200; Abcam, ab5694), antigen retrieval was performed using sodium citrate (10 mM, pH 6) before blocking.
10
Immunoperoxidase Staining of Paraffin-Embedded Tissue
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Immunoperoxidase staining was performed on paraffinembedded tissue sections, according to the protocol published elsewhere. 12 Briefly, tissue samples were deparaffinized, rehydrated, and rinsed for 10 minutes in PBS. Endogenous peroxidase was blocked for 45 minutes with 3% H 2 O 2 /methanol in the dark. Each reaction was followed by rinsing for 10 minutes in PBS. The sections were pretreated for 5 minutes with 10 mg/mL protease XXIV (Sigma-Aldrich) and chondroitinase (Sigma-Aldrich). The antibodies were applied at a dilution of 1:100 in PBS for 12 hours at room temperature. A standard peroxidaseantiperoxidase procedure was followed, and a peroxidasecoupled goateanti-rabbit (Dako) or goat anti-rat antibody (Dako) was applied at a dilution of 1:150 in PBS for 1 hour at room temperature. The color reaction was performed using a diaminobenzidine substrate (Sigma-Aldrich).
Negative controls were performed by treating the sections with swine serum instead of the primary antibody for the Laminins/Nidogens in Chondrocytes
The American Journal of Pathology -ajp.amjpathol.org polyclonal antibodies and isotype matched IgGs for the monoclonal mouse antibody. Furthermore, color reactions were performed without prior antibody treatment. All experimental data are representatives of five individual immunoreactions.
Negative controls were performed by treating the sections with swine serum instead of the primary antibody for the Laminins/Nidogens in Chondrocytes
The American Journal of Pathology -ajp.amjpathol.org polyclonal antibodies and isotype matched IgGs for the monoclonal mouse antibody. Furthermore, color reactions were performed without prior antibody treatment. All experimental data are representatives of five individual immunoreactions.
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