Alkaline Phosphatase Staining Protocol of Cells

Mammalian mulberry embryo, blastocyst cells, and ESCs express alkaline phosphatase (AKP). Differentiated embryonic cells (ECs) and ESCs are weakly positive or negative for AKP. Therefore, AKP is one of the common markers to identify the differentiation between ECs and ESCs.
AKP hydrolyzes sodium α-phosphogalactone in the incubation solution under alkaline (pH 9.0-9.6) conditions to produce α-offering phenol, which is then coupled with azo salts to produce an insoluble sunlight-resistant dye for color development.

Cells cultured in 6-well plates or other containers are taken, and the original culture medium is discarded. Wash 3 times with PBS.
Add alkaline phosphatase staining solution (azo-coupling method) and fix for 3 minutes or 4% paraformaldehyde for 10-15 minutes, and wash with PBS.
Add prepared alkaline phosphatase staining solution (azo-coupling method) dropwise. Put them into the wet box, and incubate for 15-20 minutes away from light. Wash with PBS.
Put into nuclear solid red staining solution or methyl green staining solution to re-stain for 3~5 minutes.

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Wash 3 times with PBS and photograph under a microscope.

Alkaline phosphatase staining is not done by metal precipitation to show alkaline phosphatase activity, but by azo-coupling (also called simultaneous coupling). The principle is that under alkaline conditions, the intracellular alkaline phosphatase can release phosphate with naphthol to produce a colored product with a coupled diazo salt (FBB salt). The product is localized in the cytoplasm. The alkaline phosphatase active site is blue in color and the results are more reliable than the metal salt precipitation method.
This method is generally used for alkaline phosphatase staining of paraffin sections, frozen sections, bone marrow cell smears, blood smears, etc. It is also known as the simultaneous coupling method.
The alkaline phosphatase reaction has few non-specific reactions and the results are reliable.
The reagent is stored at -20°C and protected from light for 6 months.

For research use only. Not for any other purpose.