Von kossa staining

Manufactured by Abcam

Sourced in United Kingdom

Von Kossa staining is a histological technique used to detect the presence of calcium deposits in tissue samples. The method involves the use of silver nitrate, which reacts with phosphate and carbonate groups in calcium-containing compounds, producing a black or brown precipitate. This staining process allows for the visualization and identification of calcified areas within the examined tissue.

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Lab products found in correlation

Differentation media bulletkits osteogenic, by Lonza (2 mentions) Differentiation media bulletkits adipogenic, by Lonza (2 mentions) Sudan 3 staining, by Merck Group (2 mentions) Ab124964, by Abcam (1 mentions) Hematoxylin and eosin, by Thermo Fisher Scientific (1 mentions) Ab21610, by Abcam (1 mentions) Envision system hrp, by Agilent Technologies (1 mentions) Ab92547, by Abcam (1 mentions) Optical microscope, by Nikon (1 mentions) Safranin o, by ScienCell (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Osteoimage bone mineralisation assay, by Lonza (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) β glycerophosphate, by Merck Group (1 mentions) Elipseti u microscope, by Nikon (1 mentions)

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Von Kossa staining kit Von Kossa

6 protocols using von kossa staining

Osteogenic and Adipogenic Differentiation of HASCs

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To induce osteogenic differentiation, HASCs, harvested at passage #5 and with 60–70% confluency, were cultured in Differentation Media BulletKits-Osteogenic (Lonza), according to manufacturer’s instructions. The differentiation potential for osteogenesis was assessed by mineralization of calcium accumulation on von Kossa staining (Abcam, Bristol, UK), according to producer’s instructions; moreover, we determined changes in RT-PCR expression of specific genes, namely, Secreted Phospho-Protein 1, Bone Gamma-carboxyglutamate (Gla) Protein (BGLAP), Runt-related transcription factor 2 (RUNX2), Alkaline Phosphatase, Liver/bone/kidney (ALPL). To induce adipogenic differentiation, HASCs harvested as indicated above were cultured in Differentiation Media BulletKits-Adipogenic (Lonza). The potential for adipogenic differentiation was assessed by Sudan III staining (Sigma-Aldrich), according to the manufacturer’s instructions. Changes in the expression of specific genes, markers of adipogenic differentiation, such as Peroxisome Proliferator-Activated Receptor Gamma (PPARG), Lipo-Protein Lipase (LPL), Fatty Acid Binding Protein 4 (FABP4), were also determined by RT-PCR.

Romani R., Pirisinu I., Calvitti M., Pallotta M.T., Gargaro M., Bistoni G., Vacca C., Di Michele A., Orabona C., Rosati J., Pirro M., Giovagnoli S., Matino D., Prontera P., Rosi G., Grohmann U., Talesa V.N., Donti E., Puccetti P, & Fallarino F. (2015). Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1. Journal of Cellular and Molecular Medicine, 19(7), 1593-1605.

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Histochemical and Immunohistochemical Characterization of Tissue Constructs

Cited 1 time

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Explant tissue samples were fixed in 4% paraformaldehyde, embedded in
paraffin, and histologic sections (5 μm thick) were prepared.
Representative sections were stained with hematoxylin and eosin (Thermo Fisher
Scientific, USA) and Masson’s trichrome (ScyTek Lab, USA) for trilayered
morphological assay, and with picrosirius red (ScyTek Lab, USA) and Safranin O
(ScienCell Research Lab, USA), as per manufactures’ protocol, to find the
presence of collagen and glycosaminoglycans, respectively [27 (link), 48 (link)]. In
addition, Von Kossa staining (Abcam, USA) was used to study mineral deposition
in the tissue constructs. Images were acquired with an optical microscope
(Nikon, Japan).
Additional unstained slides were prepared for immunohistochemical
analysis using antibodies against elastin (ab21610, Abcam, USA), vimentin
(ab92547, Abcam, USA), and α-smooth muscle actin (α-SMA)
(ab124964, Abcam, USA) [27 (link), 48 (link)]. Sections were blocked with the Dako
peroxidase solution, incubated with the primary antibodies overnight at 4
°C, and then with the secondary antibody (Dako Envision System HRP).
Coverslips were mounted with Permount mounting media, and images acquired using
an optical microscope (Nikon, Japan).

Jana S., Franchi F, & Lerman A. (2021). Fibrous heart valve leaflet substrate with native-mimicked morphology. Applied materials today, 24, 101112.

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Inducing Osteoblast Differentiation from iPSCs

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To induce osteoblast differentiation, iPSCs were plated (in colony pieces) at a density of 7×10⁴ cells per well of a 24-well OsteoAssay surface coated plate (Corning, Wiesbaden, Germany) in iPSC media. The following day, the media was replaced with osteogenic media [DMEM/F12, supplemented with: 15% FCS, 2 mM L-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all Invitrogen), 10 mM β-glycerophosphate, 10 nM dexamethasone and 28 µM ascorbic acid (all Sigma-Aldrich)]. Cells were cultured for 21 days with the media replaced every 2–3 days prior to analysis.
To determine matrix mineralisation, von Kossa staining (Abcam, Cambridge, UK) was carried out according to the manufacturer's instructions. Alizarin Red S staining for calcium deposition was also performed by incubating differentiated iPSCs with 2% Alizarin Red S pH 4.2 for 5 min. Hydroxyapatite deposition was detected using the OsteoImage bone mineralisation assay (Lonza, Slough, UK) according to the manufacturer's instructions. Alkaline phosphatase activity was measured using a quantitative colorimetric test on cell culture supernatant (Abcam) according to the manufacturer's instructions.

Baird A., Lindsay T., Everett A., Iyemere V., Paterson Y.Z., McClellan A., Henson F.M, & Guest D.J. (2018). Osteoblast differentiation of equine induced pluripotent stem cells. Biology Open, 7(5), bio033514.

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Osteogenic Differentiation of ASCs

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Osteogenic differentiation was conducted as we previously described with several modification (10 (link)). In details, ND-ASCs and T2DM-ASCs were seeded at a density of 2×10⁴ cells/cm² at 37℃, 5% CO₂. When cells reached 80% confluent, the supernatant was discarded and changed into osteogenic differentiation medium containing 100 nM dexamethasone, 50 μM L-ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate in Serum-free human MSC medium (Yocon). The medium was subsequently changed twice a week. After 28 days, osteogenic differentiation was assessed by von Kossa staining (Abcam). The phase contrast image was photographed with Nikon ElipseTi-U microscope (Nikon, Tokyo, Japan).

Wang L., Zhang L., Liang X., Zou J., Liu N., Liu T., Wang G., Ding X., Liu Y., Zhang B., Liang R, & Wang S. (2020). Adipose Tissue-Derived Stem Cells from Type 2 Diabetics Reveal Conservative Alterations in Multidimensional Characteristics. International Journal of Stem Cells, 13(2), 268-278.

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Osteogenic Differentiation of fHASCs

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fHASCs, harvested at passage five, were cultured in Differentation Media BulletKits-Osteogenic (Lonza), according to manufacturer's instructions. Changes in the expression of genes, markers of osteogenic differentiation, namely, secreted Phospho-Protein 1 (SPP1), Bone Gamma-carboxyglutamate (Gla) Protein (BGLAP), and Runt-related transcription factor 2 (RUNX2) were assessed by RT-q-PCR using specific primers (Table 1). The differentiation potential for osteogenesis was assessed by mineralization of calcium accumulation on von Kossa staining (Abcam). Percent cell staining was determined by measuring von Kossa-positive stained areas relative to a total selected area, in five different images, using ImageJ software (NIH, Bethesda, MD, USA).

Romani R., Manni G., Donati C., Pirisinu I., Bernacchioni C., Gargaro M., Pirro M., Calvitti M., Bagaglia F., Sahebkar A., Clerici G., Matino D., Pomili G., Di Renzo G.C., Talesa V.N., Puccetti P, & Fallarino F. (2018). S1P promotes migration, differentiation and immune regulatory activity in amniotic-fluid–derived stem cells. European Journal of Pharmacology, 833, 173-182.

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Mesenchymal Stem Cell Differentiation

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Embryoid body (EB) formation was assessed by the method described by Valli A. et al. 27 To induce osteogenic differentiation, HASCs, harvested at passage 5 and with 60-70% confluency, were cultured in Differentiation Media BulletKits-Osteogenic (LONZA), according to manufacturer's instructions. The differentiation potential for osteogenesis was assessed by mineralization of calcium accumulation on von Kossa staining (Abcam), according to producer's instructions; moreover, we determined changes in RT-PCR expression of specific genes, namely, Secreted Phospho-Protein 1 (SPP1), Bone Gammacarboxyglutamate (Gla) Protein (BGLAP), Runt-related transcription factor 2 (RUNX2), Alkaline Phosphatase, and Liver/ bone/kidney (ALPL). To induce adipogenic differentiation, HASCs harvested as indicated above were cultured in Differentiation Media BulletKits-Adipogenic (LONZA). The potential for adipogenic differentiation was assessed by Sudan III staining (Sigma-Aldrich), according to the manufacturer's instructions. The changes in the expression of specific genes, markers of adipogenic differentiation, such as Peroxisome Proliferator-Activated Receptor Gamma (PPARG), Lipo-Protein Lipase (LPL), and Fatty Acid Binding Protein 4 (FABP4), were also determined by RT-PCR.

Romani R., Fallarino F., Pirisinu I., Calvitti M., Caselli A., Fiaschi T., Gamberi T., Matino D., Talesa V.N., Donti E., Puccetti P., Modesti A, & Magherini F. (2015). Comparative proteomic analysis of two distinct stem-cell populations from human amniotic fluid. Molecular bioSystems, 11(6).

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