A segment of the abdominal aorta was excised and fixed in 10% formalin for 24 h, then dehydrated, embedded in paraffin, and cut into 5-μm -thick sections. The slides were deparaffinized, dehydrated and washed in distilled water (ddH2O),the samples were incubated with reagent A of von Kossa staining Kit (Solarbio Life Sciences, China) and placed under ultraviolet light for 10 min,and then placed into reagent B for 2 min followed by staining with hematoxylin for the demonstration of cell nucleus.
Von kossa staining kit
Manufactured by Solarbio
Sourced in China
The Von Kossa staining kit is a laboratory tool used to detect the presence of calcium deposits in tissue samples. It provides a reliable and standardized method for the visualization of mineralized structures, such as bone and cartilage, within histological preparations.
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6 protocols using von kossa staining kit
1
Aortic Calcification Evaluation
2
Aortic Mineralization Analysis in Mice
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Mice were sacrificed at 12 days after first injection. Isolated the aortas and fixed in 4% paraformaldehyde overnight at 4°C, and then put the fixed aortas into 5%, 10%, 20% and 30% sucrose solution. Embedded the aortas with OCT (#4583, Sakura, US) and cut into 10 μm section. The following staining protocol was performed by Von-kossa staining kit (#G3282, Solarbio, China). In brief, washed the sections with distilled water for 30 min, then incubated with silver nitrate solution and exposed with UV for 30min, finally treated the sections with sodium thiosulfate solution for 10 minutes at room temperature and observed under the microscope.
3
Arterial Calcification Assessment via Von Kossa
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Von Kossa staining was performed to examine calcification of the arteries according to the manufacturer’s instructions (Von Kossa staining kit, Solarbio, Beijing, China), and calcium staining of the arteries was analysed by light microscopy. Huawei P20 mobile phone was used to record images.
4
Silver Nitrate Staining of Blood Vessels
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The blood vessel slices were removed from the glass slides, treated with APES, and dried. Toluene was used twice for dewaxing for 15 min each time, and 100%, 95%, and 80% gradient ethanol was added. The slices were washed with water, and 1 mL of 1% silver nitrate solution was added. The solution was irradiated under sunlight for 30 min. The silver nitrate solution was removed and 1 mL of 5% sodium thiosulfate solution was added and left for 1 min. Basic fuchsin was used for backstaining for 10 seconds, followed by dehydration twice with 95% and absolute ethanol. The xylene was transparent after dehydration; the slides were mounted with natural resin glue, and microscopic images were obtained and analyzed.
The cells were washed with phosphate-buffered saline (PBS; pH: 7.2–7.4) and fixed with 4% paraformaldehyde for 30 min at 37 ℃. The cells were washed again with PBS, treated with reagent A in the Von Kossa staining kit (Cat. #G1452, Solarbio), and irradiated with ultraviolet light until the calcium phosphate crystals turned black. The cells were washed three times with PBS to remove excess dye. After 2 min of sodium thiosulfate treatment, the cells were washed three times with PBS. The cells were stained with eosin staining solution (Cat. #G1120, Solarbio) and observed using a microscope.
The cells were washed with phosphate-buffered saline (PBS; pH: 7.2–7.4) and fixed with 4% paraformaldehyde for 30 min at 37 ℃. The cells were washed again with PBS, treated with reagent A in the Von Kossa staining kit (Cat. #G1452, Solarbio), and irradiated with ultraviolet light until the calcium phosphate crystals turned black. The cells were washed three times with PBS to remove excess dye. After 2 min of sodium thiosulfate treatment, the cells were washed three times with PBS. The cells were stained with eosin staining solution (Cat. #G1120, Solarbio) and observed using a microscope.
5
Abdominal Aorta Histological Analysis
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A segment of the abdominal aorta was excised and fixed in 10% formalin for 24 h, then dehydrated, embedded in paraffin, and cut into 5-µm -thick sections. The slides were deparaffinized, dehydrated and washed in distilled water (ddH2O),the samples were incubated with reagent A of Von Kossa staining Kit (Solarbio Life Sciences, China) and placed under ultraviolet light for 10 min, and then placed into reagent B for 2 min followed by staining with hematoxylin for the demonstration of cell nucleus.
6
Aortic Calcification Evaluation Protocols
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After cultured at speci ed conditions for 14 days, SMCs on dishes were xed with 10% formalin for 30 min, then washed with double distilled water (ddH 2 O) twice and incubated with Alizarin Red (Solarbio Life Sciences, China) for ve minutes and washed with ddH 2 O twice to remove the excessive dye. After examination and photography under a microscopy, the dye on the cells was extracted with 100ul Hexadecyl Pyridinium chloride (Sigma-Aldrich) and the optical density (OD) at 560 nm was measured using a microplate reader (Spectra MAX 190, Molecular Devices, USA). von Kossa staining A segment of the abdominal aorta was excised and xed in 10% formalin for 24 h, then dehydrated, embedded in para n, and cut into 5-µm -thick sections. The slides were depara nized, dehydrated and washed in distilled water (ddH 2 O),the samples were incubated with reagent A of von Kossa staining Kit (Solarbio Life Sciences, China) and placed under ultraviolet light for 10 min ,and then placed into reagent B for 2 min followed by staining with hematoxylin for the demonstration of cell nucleus.
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