Cryosections (10 μm) from T1 and T4 3D pellets were incubated for 1 h at 4 °C with primary antibodies against COLII (1:200 dilution, MAB 1330, Ms, Millipore) and SOX9 (1:500 dilution, AB5535, Rb, Abcam). Antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 555 (Thermo Fisher Scientific) were used as secondary antibodies for COLII and SOX9, respectively. Sections were mounted in mounting medium (Dako Cytomation) and visualized on an LSM 880 Meta confocal microscope. Fluorescence was monitored in the appropriate channels to detect Alexa Fluor 555 (1:1000 dilution, A21429, excitation, 555 nm; emission, 565 nm), Alexa Fluor 488 (1:1000 dilution, A21202, excitation, 495 nm; emission 519 nm), and DAPI (excitation, 358 nm; emission, 461 nm).
Mab1330
Manufactured by Merck Group
Sourced in United States, United Kingdom
MAB1330 is a laboratory equipment product. It is designed to perform a specific core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Alcian blue 8gx, by Merck Group (2 mentions)
Ab34710, by Abcam (2 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab5535, by Abcam (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
3 diaminobenzidine dab solution, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Avidin biotin peroxidase complex detection kit, by Agilent Technologies (1 mentions)
Papain, by Merck Group (1 mentions)
Tcs lsi laser confocal microscope, by Leica (1 mentions)
Quant it picogreen kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
6 protocols using mab1330
1
Immunostaining of Chondrocyte Markers
2
Chondrogenesis Histological Analysis
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Following chondrogenesis, pellets were fixed in 4% formaldehyde and embedded in paraffin. After sectioning, deparaffinization and rehydration sections were analysed by histology [1% Alcian Blue 8-GX (Sigma-Aldrich, Zwijndrecht, The Netherlands)] and immunohistochemistry [COL2 (MAB1330 1:100; Merck-Milipore, Zwijndrecht, The Netherlands), COL1 (ab34710 1:100; Abcam, Amsterdam, The Netherlands) and OPG (EPR3592 1:100; Epitomics-Abcam, Amsterdam, The Netherlands)], as described before [12 (link)]. Pixel intensity quantification was performed for Alcian Blue staining by ImageJ, and surface area of the pellets were measured with the CellSens Dimension software (Olympus, Leiderdorp, The Netherlands).
3
Histological and Immunohistochemical Analysis of Engineered Cartilage and Bone
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Tissues (neo-cartilage and neo-bone) were fixed in 4% formaldehyde and embedded in paraffin. After sectioning, slides were deparaffinized and rehydrated prior to histology or immunohistochemistry.
Overall cellular and tissue structure was visualized with hematoxylin–eosin (HE) staining. Glycosaminoglycans were visualized by staining with 1% Alcian Blue 8-GX (Sigma-Aldrich) and Nuclear Fast red staining (Sigma-Aldrich). Calcium deposits were stained with 2% Alizarin Red S (Sigma-Aldrich).
To detect COL2 (MAB1330; Millipore; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), COL1 (ab34710; Abcam; 1:1000 in TBST/10% normal goat serum, overnight at 4 °C), and COL10 (× 53/2031501005; Quartett; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), immunohistochemistry was performed with 3-diaminobenzidine (DAB) solution (Sigma-Aldrich) and hematoxylin (Klinipath) as described before (Bomer et al. 2015 (link)).
Lipid droplets were stained for 10 min with Oil-Red-O solution (Sigma-Aldrich) after fixation of the cells in 4% formaldehyde. To reduce the background, the following staining cells were gently washed with 60% isopropanol and distilled water.
Overall cellular and tissue structure was visualized with hematoxylin–eosin (HE) staining. Glycosaminoglycans were visualized by staining with 1% Alcian Blue 8-GX (Sigma-Aldrich) and Nuclear Fast red staining (Sigma-Aldrich). Calcium deposits were stained with 2% Alizarin Red S (Sigma-Aldrich).
To detect COL2 (MAB1330; Millipore; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), COL1 (ab34710; Abcam; 1:1000 in TBST/10% normal goat serum, overnight at 4 °C), and COL10 (× 53/2031501005; Quartett; 1:100 in TBST/10% normal goat serum, overnight at 4 °C), immunohistochemistry was performed with 3-diaminobenzidine (DAB) solution (Sigma-Aldrich) and hematoxylin (Klinipath) as described before (Bomer et al. 2015 (link)).
Lipid droplets were stained for 10 min with Oil-Red-O solution (Sigma-Aldrich) after fixation of the cells in 4% formaldehyde. To reduce the background, the following staining cells were gently washed with 60% isopropanol and distilled water.
4
Immunolocalization of Type II Collagen
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Immunolocalization of collagen type II was performed in fixed samples. Constructs were permeabilized with 0.2% Triton X100 (Sigma, X100) in PBS for 5 min and then it was blocked with a solution of 3% BSA (Sigma, A2153) in PBS for 30 min. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide solution during 30 min. Samples were rinsed in PBS during 5 min. R.T.U. Vectastain 1 Universal Elite ABC Kit (Vector/VCPK-7200) was used for antibody incubation, according to the instructions of the manufacturer. Shortly, samples were incubated with anti-human collagen type II (Millipore Iberica/ MAB1330) overnight at 4 C, in a humidified atmosphere. Incubation was revealed by using the Peroxidase Substrate Kit DAB (Vector/VCSK-4100), according to the instructions of the manufacturer. Samples were washed in water during 5 min and counterstained with hematoxilin for nuclei visualization. Finally, slides were mounted in Entellan rapid.
5
Immunohistochemical Analysis of Chondrogenic Markers
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Cultured ADSCs, chondrocytes and dPNCG, the 3D sample was sectioned into 5 μm sections and fixed on coated glass slides. Preheated 10 mM citrate buffer (pH 6.0) at 100 °C for 30 min for antigen retrieval was done. Immunostaining was done with a standard avidin-biotin-peroxidase complex detection kit (DakoCytomation, Glostrup, Denmark) according to the instructions for use. Primary antibodies were incubated for 60 minutes at room temperature, anti-aggrecan antibody, GTX54920, 1:100 dilutions, GeneTex, Inc. USA; anti-collagen II antibody, MAB1330, 1:100 dilutions, Merck, USA and secondary antibody Goat Anti-Rabbit IgG-Biotin, ab6720, 1:3000 dilutions, Abcam, Cambridge, MA, USAGoat Anti-Mouse IgG1-Biotin, ab98691, 1:2000 dilutions, Abcam, Cambridge, MA, USA, followed by the biotinylated secondary antibody for 1 h, and peroxidase-conjugated streptavidin for 30 min before washing with TBST three times for 5 min. The sections were covered with the DAB solution for 5 min to localize positive staining and counterstained with hematoxylin for 1 min, mounted and photographed and semi-quantified by ImageJ software (Wayne Rasband, National Institute of Health, USA).
6
Characterization of MSC-Seeded Scaffolds
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MSC-seeded scaffolds were harvested at different cultivation times (day 1, 7, 14, and 28), washed with PBS and individually digested 500 µl papain digestive solution (280 µg/ml papain, 50 mM EDTA, 5 mM L-cysteine in Dulbecco's PBS pH 6.5, all from Sigma-Aldrich) at 45 o C for 2 days. The supernatants of the digested samples were used for monitoring total DNA content using a Quant-iT TM Picogreen® kit (Invitrogen). Dimethymethylene blue (DMMB, Sigma-Aldrich) and a hydroxyproline assay kit (QuickZyme Bioscience, UK) were used to measure the amount of secreted sulfated glycosaminoglycan (sGAG) and total collagen, respectively. All samples and standards were done in triplicate. Immunostaining of type II collagen was performed using primary antibody (MAB1330, Merck Millipore, UK) and secondary antibody labelled with Alexa Fluor 488 (Abcam, ab150113). The sections were counterstained with Hoechst 33258 and imaged using a Leica TCS LSI laser confocal microscope.
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