4.5.1 Alkaline phosphatase staining
Tokyo Institute of Technology
18.104.22.168 General information
Endothelial cells possess an endogenous alkaline phosphatase (AP) activity.
The enzymatic activity of AP is not restricted to the endothelial lineage. In medaka embryos, somite cells during early developmental stages and head mesenchymal cells during later stages are AP positive. The vascular AP activity can be detected from stage 23 to the adult stage.
- Fix embryos at required stages with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4¡î over night.
- Dechorionate embryos.
- Remove PFA and wash twice with PBS + 0.1% Tween-20 (PBT) for 5min at room temperature (RT).
- Wash in 50% methanol / 50% PBT for 15min at RT.
- Wash in 100% methanol twice for 15min at RT.
- Store at -20¡î over night (if necessary up to 3 days)
- Rinse twice with PBT for 5 min at RT.
- Equilibrate embryos with NTMT (100mM Tris pH 9.5, 100mM NaCl, 50mM MgCl2, 0.1% Tween-20) with 3rinses (5 min twice and 30 min ) at RT.
- Start the color reaction by incubating in staining solution (168mg 4-Nitroblue tetrazolium (NBT) and 84.5 mg 5-Bromo-4-chloro-8-indolilphosphate (BCIP) per 1 ml of NTMT.
- Stop after 40-120 min by washing with PBT 3 times.
- Transfer embryos into 70 % glycerol (if necessary).
This protocol is modified for medaka embryos from the protocol kindly given by Dr Torsten Trowe.