Samplepreparation Washembryos1xin25%MMR Removeexcessbuffer Add10volumes"incubationbuffer",i.e.50µlfor5embryos LysetheembryosbygentlypipettingupanddownusingalargebluetipUseanegativecontrol,asshearingofgenomicDNAmaygivefalsepositivesignals Dilute10µllysatewith190µl"incubationbuffer"Considertestingotherdilutions Incubatethissamplefor30min.at4°C Centrifugeat13,000rpm(Eppendorf)for10min.at4°C Remove160µlsupernatant/embryo,avoidingpelletcontaminationofthesupernatant Samplescanbestored,ifnotusedthesameday,inaliquotsat-20°C DoELISAaccordingtomanufacturer"sinstructions.