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Alkaline phosphatase staining

4.5.1.1Generalinformation

Endothelialcellspossessanendogenousalkalinephosphatase(AP)activity.TheenzymaticactivityofAPisnotrestrictedtotheendotheliallineage.Inmedakaembryos,somitecellsduringearlydevelopmentalstagesandheadmesenchymalcellsduringlaterstagesareAPpositive.ThevascularAPactivitycanbedetectedfromstage23totheadultstage.

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4.5.1.2Preparation

  • Fixembryosatrequiredstageswith4%paraformaldehyde(PFA)inphosphatebufferedsaline(PBS)at4¡îovernight.
  • Dechorionateembryos.
  • RemovePFAandwashtwicewithPBS+0.1%Tween-20(PBT)for5minatroomtemperature(RT).
  • Washin50%methanol/50%PBTfor15minatRT.
  • Washin100%methanoltwicefor15minatRT.
  • Storeat-20¡îovernight(ifnecessaryupto3days)
    • ↑

    4.5.1.3Staining

  • RinsetwicewithPBTfor5minatRT.
  • EquilibrateembryoswithNTMT(100mMTrispH9.5,100mMNaCl,50mMMgCl2,0.1%Tween-20)with3rinses(5mintwiceand30min)atRT.
  • Startthecolorreactionbyincubatinginstainingsolution(168mg4-Nitrobluetetrazolium(NBT)and84.5mg5-Bromo-4-chloro-8-indolilphosphate(BCIP)per1mlofNTMT.
  • Stopafter40-120minbywashingwithPBT3times.
  • Transferembryosinto70%glycerol(ifnecessary).
    • ↑

    4.5.1.4Reference

    ThisprotocolismodifiedformedakaembryosfromtheprotocolkindlygivenbyDrTorstenTrowe.


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