AP staining was performed using the FastRed Alkaline Phosphatase Kit (Sigma). ESCs were fixed in ice-cold 4% PFA for 1 min. ESCs were subsequently washed and incubated with staining solution for 15 min at room temperature followed by examination under the microscope to capture images.
Fastred alkaline phosphatase kit
Manufactured by Merck Group
The FastRed Alkaline Phosphatase Kit is a laboratory product used to detect and quantify alkaline phosphatase activity. It provides a sensitive and specific method for measuring this enzyme, which is commonly used as a biomarker in various applications.
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2 protocols using fastred alkaline phosphatase kit
1
Alkaline Phosphatase Staining of ESCs
2
Immunostaining for Gata4 expression
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AP staining was carried out using the FastRed Alkaline Phosphatase Kit (Sigma) according to the manufacturer's protocol.
In the immunostaining assay, the cells were washed with PBS, fixed with 4% paraformaldehyde (4% PFA) at room temperature for 20 min and then permeabilized with 0.2% Triton X-100 for 8 min. The cells were blocked for 1 h in 10% FBS (v/v in PBS), incubated overnight with primary antibodies (anti-Gata4; Santa Cruz Biotechnology) at 4°C, and then washed three times with 10% FBS (v/v in PBS). Next, the cells were stained with fluorescent secondary antibodies in the dark for 1 h and counterstained with Hoechst 33342 for 10 min at room temperature. Finally, the cells were examined under a fluorescence microscope to acquire fluorescent images.
In the immunostaining assay, the cells were washed with PBS, fixed with 4% paraformaldehyde (4% PFA) at room temperature for 20 min and then permeabilized with 0.2% Triton X-100 for 8 min. The cells were blocked for 1 h in 10% FBS (v/v in PBS), incubated overnight with primary antibodies (anti-Gata4; Santa Cruz Biotechnology) at 4°C, and then washed three times with 10% FBS (v/v in PBS). Next, the cells were stained with fluorescent secondary antibodies in the dark for 1 h and counterstained with Hoechst 33342 for 10 min at room temperature. Finally, the cells were examined under a fluorescence microscope to acquire fluorescent images.
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