Histochemical staining of sea
urchin embryos for alkaline phosphatase (AP) enzyme
activity
1.
Obtain embryo samples, tube of AP
substrate buffer and tube of
phosphate buffered saline (PBS)
for each group. Allow embryos to settle. Carefully remove
supernatant.
2.
Resuspend in 0.5 ml AP substrate buffer. Allow embryos to
settle for 10 min. Remove excess buffer.
3. Add
100 ul AP substrate to tubes. Check for staining after 5
minutes by transferring a small sample to depression slide
and observing on 4X or by observing tube of embryos using
dissecting microscope. Be careful not to get AP
substrate on your hands (wear gloves) or on your microscope
. Do not leave light turned on between observations. To
stop the reaction, return embryos to the tube and add 0.5 ml
PBS.
4.
Allow embryos to settle for 10 minutes. Remove buffer to
about 100 ul, return to depression slides and observe. Look
for evidence of morphogenesis (archenteron invagination) and
tissue differentiation (gut alkaline phosphatase activity
and spicule formation). Document your observations by
capturing images of your stained embryos.
Alkaline Phosphatase substrate: Western Blue
stabilized substrate (Promega)
|