The following protocol is for detection of human placental alkaline phosphatase (AP) in cultured cells. Human placental AP is heat stable, unlike other AP proteins typically expressed by cells. A few cell types (HeLa cells, for example) express high background levels of heat-stable AP making detection of introduced AP difficult, but most cells have suitably low AP background.
1. FIX: Aspirate culture medium and fix cells in 0.5% glutaraldehyde (or 2-4% paraformaldehyde) in phosphate buffered saline (PBS) for 5-10 min. It is not necessary to wash the cells with PBS prior to fixation.
2. WASH: Wash cells 3 times with PBS.
3. HEAT: Heat cells at 65°C for 30 min in PBS to inactivate heat-labile AP in cells. Human placental AP is heat stable to at least 68°C. Do not heat in AP staining buffer below as it can destroy cell morphology. Cells can be washed with AP buffer after heating and prior to staining to speed the staining rate (phosphate in PBS can inhibit XPHOS dephosphorylation by AP).
4. STAIN: Prepare fresh staining solution by adding the XPHOS and NBT stock solutions to the AP stain buffer at 1X concentrations. Avoid exposure of staining solution to light. Stain cells for 30 min to overnight in the dark. Wash off staining solution with water.
XPHOS - 100X solution is 10 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, disodium salt (Sigma) in water, store at -20°C.
NBT - 100X solution is 50 mg/ml Nitro Blue tetrazolium (Sigma) in 70% dimethylformamide:30% water. Store at -20°C in glass covered with aluminum foil to prevent destruction by light.
AP BUFFER - 100 mM Tris pH 8.5, 100 mM NaCl, 50 mM MgCl2.
NOTE: Cells can be stained for beta-galactosidase using Xgal (5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside) and then stained for AP. Wash cells thoroughly with PBS before staining for AP.