AP staining was performed using AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma-Aldrich) according to the manufacturer's instructions.
Ast fast red tr
Manufactured by Merck Group
Sourced in United States
The AST Fast Red TR is a laboratory reagent used in various analytical and diagnostic applications. It is a diazonium salt that can be used as a chromogenic substrate in enzyme-linked immunosorbent assays (ELISA) and other colorimetric assays. The AST Fast Red TR provides a characteristic red color change when reacted with the appropriate enzyme or substrate, enabling the detection and quantification of target analytes.
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Lab products found in correlation
8 protocols using ast fast red tr
1
Alkaline Phosphatase Staining Protocol
2
Alkaline Phosphatase Activity Assay in piPSCs
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The alkaline phosphatase (AP) activity of piPSCs was determined by AST Fast Red TR (Sigma-Aldrich) and a-Naphthol AS-MX Phosphate (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, cells were washed twice with ice-cold PBS, fixed with 4% paraformaldehyde (pH 7.4) for 15 min at room temperature, and followed by washing three times with ice-cold PBS. Then the cells were then incubated at room temperature with dye solution containing AST Fast Red TR (1.0 mg/ml), a-Naphthol AS-MX (0.4 mg/ml) in 0.1 M Tris Buffer. After 10–20 min incubation, the cells were washed three times with PBS and the images were documented with a Nikon microscope.
3
Alkaline Phosphatase Staining of piPSCs
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Suitable piPSCs were fixed with 4% paraformaldehyde (pH 7.4) for 15 min, then washed 2–3 times with PBS. The cells were stained with AST Fast Red TR and α-naphthol AS MX phosphate (Sigma-Aldrich, USA) according to the manufacturer’s instructions. In brief, 1.0 mg/mL AST Fast Red TR, 0.4 mg/mL α-naphthol AS MX phosphate, and 0.1 mmol/L Tris-HCL 8.8 buffers were used to prepare the AP staining solution. The cells were incubated for 20 min with AP stain, which was then replaced with PBS. The AP-positive piPSC clones showed red and images were captured using a phase contrast microscope (Nikon, Japan).
4
Alkaline Phosphatase Staining of piPSCs
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The piPSCs (cultured for 5 days) were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 15 min, with AST Fast Red TR and α-naphthol AS MX phosphate (Sigma-Aldrich, USA) then used to stain the cells according to the manufacturer’s instructions. The piPSCs were incubated in 1.0 mg/mL Fast Red TR, 0.4 mg/mL α-naphthol AS-MX, and 0.1 mmol/L Tris-HCL 8.8 buffer at room temperature for 20 min. The AP-positive piPSCs clones showed red (Zhang et al., 2017 (link)). Images were obtained using a Nikon phase difference microscope.
5
Measuring Alkaline Phosphatase Activity
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Stem cells show high alkaline phosphatase (AP) activity, which decreased after differentiation. In this study, AP staining was used to measure AP activity. Briefly, AST Fast Red TR (F6760, Sigma Aldrich) and α-Naphthol AS-MX Phosphate (N4875-1G, Sigma Aldrich) were used to detect AP activity. Ice-cold PBS (SH30256.01B, Hyclone Laboratories) was used to wash cells twice; after being fixed with 4% paraformaldehyde, the cells were then incubated with the mixture of Fast Red TR and Naphthol AS-MX for 10 min.
6
Immunostaining of Induced Pluripotent Stem Cells
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Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 minutes at room temperature, washed twice using ice‐cold PBS and developed with AST Fast Red TR and α‐Naphthol AS‐MX Phosphate (Sigma‐Aldrich) according to the manufacturer's instructions. Then the cells were incubated with the mixture (1.0 mg/mL Fast Red TR, 0.4 mg/mL Naphthol AS‐MX in 0.1 mol/L Tris‐HCL Buffer) at room temperature. After 20 minutes, the AP‐positive iPS colonies showed in red colour. The images were documented by a Nikon phase contrast microscope.14
7
Alkaline Phosphatase Activity Staining
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Alkaline phosphatase (AP) activity was detected by using AST Fast Red TR (Sigma) and α‐Naphthol AS‐MX Phosphate (Sigma) in accordance with the manufacturer's protocol. Cells were washed with phosphate‐buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. The fixed cells were washed once with PBS and incubated with the mixture at room temperature for 15 minutes.15 The cells were observed and the images were captured by using a Nikon (Tokyo, Japan) inverted microscope after staining.
8
Alkaline Phosphatase Assay for iPSCs
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AP activity of piPSCs was detected by AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma Aldrich) according to the manufacturer’s instructions. Briefly, cells were washed twice using ice-cold PBS, and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, and washed three times with ice-cold PBS. The cells were then incubated with the mixture (1.0 mg/mL Fast Red TR, 0.4 mg/mL Naphthol AS-MX in 0.1 M Tris Buffer) at room temperature. After 10 min incubation, the AP-positive iPS colonies showed in red color. The images were documented by a Nikon fluorescence microscope.
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