The in situ detection of enzyme alkaline phosphatase was performed at 3, 7, and 10 days. After removing the culture medium, the wells were washed twice with PBS (Phosphate buffered saline) solution heated to 37 °C. Then, three hundred and twenty milligrams of the Triz reagent (Sigma) were dissolved in 20 mL of deionized water and, added, 7 mg of the Fast Red reagent (Sigma). 2 mL of this solution was discarded and 8 mg of naphthol (Sigma) diluted in 2 mL of dimethylformamide (Merck) were added to form the working solution. One milliliter of this solution was added to each well. The plate was then taken to the incubator in a humidified atmosphere at 37 °C with 5% of CO2 for 30 minutes. The solution was then taken out of the wells, and the plate was allowed to dry at room temperature before being subjected to qualitative and quantitative examination using Image J.
Fast red reagent
Manufactured by Merck Group
The Fast Red reagent is a laboratory product manufactured by Merck Group. It is a chromogenic substrate used in various laboratory applications to detect the presence of specific target molecules or enzymatic activities. The Fast Red reagent produces a red-colored reaction product when it interacts with the target analyte, enabling visual or colorimetric detection.
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Lab products found in correlation
4 protocols using fast red reagent
1
In Situ Alkaline Phosphatase Detection
2
In situ Alkaline Phosphatase Detection
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In situ ALP detection was also performed after 10 days. Cells were washed twice with PBS at 37°C. Then, 320 mg of Trizma® Pre‐set crystals (Sigma‐Aldrich) were dissolved in 20 mL of deionized water. From this solution, 18 ml were added to 2 mL of dimethylformamide (Merck) with 8mg Naphthol (Sigma‐Aldrich). This was followed by addition of 7mg of Fast Red reagent (Sigma‐Aldrich) to the final work solution. Finally, cells were incubated in 1 mL of this solution in a humidified atmosphere at 37°C with 5% CO2 for 30 minutes and then removed from the wells for subsequent analysis.
3
In situ Alkaline Phosphatase Detection
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Analysis of in situ detection of alkaline phosphatase was performed after 7, 10 and 14 days. Cells were washed twice with PBS at 37 °C. There were dissolved 320 mg of Trizma® Pre-set crystals (Sigma-Aldrich) in 20 mL of deionized water. From this solution, 18 mL were added to 2 mL of dimethylformamide (Merck) with 8 mg Naphthol (Sigma-Aldrich). The next step was adding 7 mg of Fast Red reagent (Sigma-Aldrich) to this final work solution. Finally, cells were incubated in 1mL of this solution in a humidified atmosphere at 37 °C with 5% CO 2 for 30 min and then removed from the wells for subsequent qualitative and quantitative analysis.
4
In Situ Alkaline Phosphatase Detection
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Detection of in situ alkaline phosphatase (ALP) was performed at 3, 7 and 10 days. After removal of the culture medium, the wells were washed twice with PBS at 37ºC. Then 320 mg of Triz reagent (Sigma) was dissolved in 20 mL of deionized water, and both 7 mg of Fast Red reagent (Sigma) and 8 mg of Naphthol (Sigma) diluted in 2 mL of dimethylformamide (Merck) were added. Each well received 1 mL of this working solution, and the plate was taken into the incubator in a humidified atmosphere with 5% CO 2 at 37ºC for 30 minutes. After this time, the solution was removed from the wells, and the plate was left to dry at RT for further photographic documentation.
The colored images were converted into binary images using Image J software, and then converted to grayscale, to allow the pixels to be counted for a quantitative analysis.
The colored images were converted into binary images using Image J software, and then converted to grayscale, to allow the pixels to be counted for a quantitative analysis.
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