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Von kossa staining

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Von Kossa staining is a histochemical technique used to detect the presence of calcium deposits in biological samples. The staining process involves the treatment of the sample with silver nitrate, which reacts with the calcium to form silver phosphate or silver carbonate. These silver compounds are then reduced to metallic silver, resulting in a black or brown staining of the calcium-containing areas within the sample. This method is commonly used in the analysis of bone, cartilage, and other mineralized tissues.

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11 protocols using von kossa staining

1

Quantifying Aortic Calcium Deposition

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Calcium deposits in aortic sections were stained by Alizarin Red S (Sigma Aldrich, USA) or Von Kossa staining (Sigma Aldrich, USA). Descending aortas were decalcified with 0.5 mmol/L hydrochloric acid overnight. Calcium released from the aortic tissues was determined colorimetrically using a calcium diagnostic kit (BIOSINO, China). The amount of vascular calcium was normalized to the weight of the tissues and expressed as μg/mg.
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2

Multilineage Differentiation Evaluation

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After ∼21 days of differentiation, the cells were fixed for cytochemical staining. Lipid droplets were visualized by using Oil Red O staining (Sigma-Aldrich), proteoglycans accumulation was visualized by Alcian Blue staining (Sigma-Aldrich) and calcium accumulation was visualized by using Von Kossa staining (Sigma-Aldrich) for adipogenic, chondrogenic and osteogenic differentiation respectively. The cells were also analysed by using quantitative RT-PCR (qRT-PCR). Total RNA was extracted by using Trizol (Invitrogen) and reverse-transcribed into cDNA by using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The qRT-PCR mixture contained cDNA, forward and reverse primers, and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The reactions were conducted by using AbiPrism 7000 Sequence Detection System (Applied Biosystems) with initial enzyme activation at 95°C for 10 min., followed by 45 cycles of denaturation at 95°C for 15 sec. and annealing and extension at 60°C for 60 sec. The expression level of genes of interest was normalized against housekeeping gene GAPDH. The fold change was calculated by using the equation 2−ΔΔCT. Primer sequences are presented in Table S1.
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3

Adipogenic and Osteogenic Differentiation of BMSCs

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For adipogenic differentiation, 1 × 105 BMSCs were seeded on 6-well plates and cultured for 14 days with adipogenic induction medium containing 1 µmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine and 100 µmol/L indomethacin. Cells cultured in MEMα medium supplemented with 15% FBS served as a negative control. Adipogenic differentiation was assessed by Oil-Red-O staining (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer instructions and by real-time PCR analysis of PPAR-γ1 and PPAR-γ2 gene expression.
For osteogenic differentiation, BMSCs were seeded at 5 × 103/cm2 on 12-well plates and cultured for 21 days in osteogenic induction medium containing 0.1 µmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 200 µmol/L ascorbate-2-phosphate. Cells cultured in MEMα medium supplemented with 15% FBS were used as a negative control. Osteogenic differentiation was detected by von Kossa staining (Sigma Aldrich) according to the manufacturer instructions and by real-time PCR analysis of osteopontin and Runx2 gene expression.
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4

Histological Analysis of Tissue Samples

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After fixation of the constructs in 10% neutral buffered formalin at room temperature for 20 min and 5 times washing with UPW, samples were dehydrated and impregnated with paraffin over night by use of a tissue processor (TPC 15 Duo, Medite AG, Winter Garden FL, United States) before being embedded in paraffin using a paraffin embedding station (TES 99, Medite AG, Winter Garden, FL, USA). Paraffin sections of 6–7 µm (HM355S, Microm International, Walldorf, Germany) were stained with Haematoxylin and Eosin (H&E) (Sigma-Aldrich, Buchs SG, Switzerland) for a general overview. Collagen distribution was visualised by Sirius Red staining while mineralisation was shown with von Kossa staining (Sigma-Aldrich, Buchs SG, Switzerland).
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5

Mineral Deposition Quantification in Chondrocytes

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Von Kossa staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to determine the extent of minerals deposited on chondrocyte cells. Silver nitrate solution was added to the cells and the plate was exposed to ultraviolet light for 30 min. Cells were subsequently washed with PBS and the reaction was stopped with the addition of 500 µl of 5% sodium thiosulfate (Sigma-Aldrich; Merck KGaA). The positive mineral depositions were stained in black. Cells were counterstained with nuclear fast red for 5 min at room temperature to label the nucleus and cytoplasm. Images were captured using an inverted microscope (magnification, ×100; Nikon Eclipse TC 100; Nikon Corporation).
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6

Multilineage Differentiation of PDLSCs

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To study PDLSC multi-differentiating capabilities, the cells were seeded in different media. Osteogenic medium (OM) was composed of α-MEM supplemented with 10% FBS, 50 μg/mL ascorbic acid, 10−8 M dexamethasone, and 10 mM beta-glycerophosphate (Sigma Aldrich, Burlington, MA, USA) and cultures were maintained for 30 days. Then, the formation of a mineralized matrix was assessed by Von Kossa staining (Sigma Aldrich, Burlington, MA, USA). Adipogenic and chondrogenic media were purchased from Miltenyi (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultures were maintained for 21 days. The formation of adipocytes were detected by oil red O staining, which colors lipid droplets. Chondrogenic differentiation was detected by Aggrecan (ACAN) staining of chondrocyte micromasses.
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7

Characterizing Osteogenic Mineralization

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After incubation for 14 days, Alizarin red staining (ARS, Thermo Fisher, Waltham, MA, United States) and von Kossa staining (Sigma-Aldrich, St. Louis, MO, United States) were performed to evaluate mature mineralized nodules during late phase of osteogenic differentiation (Kwon et al., 2014 (link)). For ARS staining, OB cells subjected to the various studied treatments were washed using DPBS, fixed in 2% paraformaldehyde, and then stained with ARS working solution. Culture plates were imaged via an optical microscope. Mineralized nodules are shown as a dark red center and light red peripheral area. For von Kossa staining, OB cells were rinsed with DPBS and fixed in 2% paraformaldehyde for 15 min, followed by staining with a freshly prepared 5% silver nitrate solution for 40 min under UV exposure. After that, cells were immersed in fresh 5% sodium carbonate to indicate minerals and matrix of calcium deposits. Lastly, positive calcium deposits with brownish-blackish color were photographed using an optical microscope.
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8

Multilineage Differentiation of Stem Cells

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Adipogenic, chondrogenic, and osteogenic differentiation of SHED and ASC were carried out as previously described at the third passage [19 (link)]. The cultures were initiated at a density of 1000 cells/cm2 in six-well plates and grown until confluence and then subjected to differentiation into adipogenic, chondrogenic, and osteogenic lineages. Briefly, the adipogenic lineage was initiated by inducing the cells with 10% FBS, 200 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine (IMBX), 10 μg/mL insulin, and 1 μM dexamethasone (all from Sigma-Aldrich). Lipid droplets were visualized using oil red staining (Sigma-Aldrich). For chondrogenic differentiation, cells were cultured in medium supplemented with ITS+1 (Sigma-Aldrich), 50 μM L-ascorbic acid 2-phosphate, 55 μM sodium pyruvate (Invitrogen), 25 μM L-proline (Sigma-Aldrich), and 10 ng/mL transformation growth factor-β (TGF-β; Sigma-Aldrich). Assessment of proteoglycan accumulation was visualized by Alcian Blue staining (Sigma-Aldrich). Osteogenic differentiation was stimulated in a 3-week culture period in medium supplemented with 10% FBS, 10–7 M dexamethasone, 10 mM glycerol phosphate (Fluka, Buchs, Switzerland), and 100 μM L-ascorbic acid 2-phosphate. The assessment of calcium accumulation was visualized using von Kossa staining (Sigma-Aldrich).
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9

Quantification of Osteoblast Mineralization

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Von Kossa staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to determine the extent of minerals deposited on osteoblast cells. Silver nitrate solution (0.5%) was added onto the cells and the plate was exposed to ultraviolet light for 30 min. Subsequently, the cells were washed with saline and the reaction was stopped with the addition of 500 µl 5% sodium thiosulfate (Sigma-Aldrich; Merck KGaA). Positive mineral depositions were stained in black. Cells were counterstained with nuclear fast red at room temperature for 5 min to label the nucleus and cytoplasm of cells. Images were obtained using an inverted microscope (Nikon Eclipse TC 100) at ×100 magnification.
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10

von Kossa Staining of Mineralized PBS Scaffolds

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von Kossa staining (Sigma-Aldrich Corporation, Germany) procedure was used to determine the extent of minerals deposited on PBS scaffolds. Mineralization of pDGSCs seeded on unmodified and fibronectin and laminin modified PBS scaffolds and TCP at the end of 10 and 20 days of incubation was evaluated. In this method, silver nitrate solution (500 μL) (Sigma-Aldrich Corporation, Germany) was added onto PBS scaffolds and the plate was exposed to ultraviolet light for 30 min. Positively charged silver ions reacted with negatively charged phosphates and carbonates in calcium deposits and were then reduced to black metallic silver by UV light. After that, the scaffolds were washed with physiological saline solution and the reaction was stopped with the addition of 500 μL of 5% sodium thiosulfate (Sigma-Aldrich Corporation, Germany). After scaffolds were washed with physiological saline solution, their images were obtained by inverted microscope (Nikon Eclipse TC 100, USA). Ultimately, cells were stained with nuclear fast red to label the nucleus and cytoplasm of cells to make cells apparently visible for brightfield microscopy.
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