Histochemicalstainingofseaurchinembryosforalkalinephosphatase(AP)enzymeactivity

1.Obtainembryosamples,tubeofAPsubstratebufferandtubeofphosphatebufferedsaline(PBS)foreachgroup.Allowembryostosettle.Carefullyremovesupernatant.

2.ResUSPendin0.5mlAPsubstratebuffer.Allowembryostosettlefor10min.Removeexcessbuffer.

3.Add100ulAPsubstratetotubes.Checkforstainingafter5minutesbytransferringasmallsampletodepressionslideandobservingon4Xorbyobservingtubeofembryosusingdissectingmicroscope.BecarefulnottogetAPsubstrateonyourhands(weargloves)oronyourmicroscope.Donotleavelightturnedonbetweenobservations.Tostopthereaction,returnembryostothetubeandadd0.5mlPBS.

4.Allowembryostosettlefor10minutes.Removebuffertoabout100ul,returntodepressionslidesandobserve.Lookforevidenceofmorphogenesis(archenteroninvagination)andtissuedifferentiation(gutalkalinephosphataseactivityandspiculeformation).Documentyourobservationsbycapturingimagesofyourstainedembryos.AlkalinePhosphatasesubstrate:WesternBluestABIlizedsubstrate(Promega)

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