Thesemethodsweresuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy. LCMandsubsequentmolecularanalysiscanbecarriedoutonslidesstainedusingstandardhematoxylinandeosinmethods.However,ifcelltypesthatare(orarenot)expressingaspecificproteinarerequiredforastudythenmoreadvancedslidepreparationmethodssuchasImmuno-LCMmaybeutilized. 1.Materials Important:Forallproteinanalysis,dissolve1proteaseinhibitorcocktailtabletper10mlofeachreagentexceptthexylenes. 2.StorageofSections 3.Methods TIP:Usetheminimalamountofstainingtovisualizethetissueformicrodissection.Thiswillsignificantlyimprovemacromoleculerecovery.Forexample,hematoxylinandeosincanbeusedat10%oftheirstandardconcentrations.Sincetheslidesaremicrodissectedwithoutacoverslip,thetissueisnotindex-matchedandsubstantiallightscatteringoccurs,typicallyproducing"dark"images.Thus,bothimagequalityandmolecularrecoverycanbeimprovedbydecreasingstainconcentrations.A:Paraffin-embeddedSectionsorFrozenSections Placethesectionsinthefollowingsolutions: B:Low-meltPolyester-embeddedSections TIP:Proceedgentlywhenstainingsectionsembeddedinpolyesterwax.Eventhoughthesectionsareplacedonchargedslides,thetissuehasatendencytodetachfromtheslideandshouldbemonitoredcarefullythroughoutthestainingprocedure. Placethesectionsinthefollowingsolutions: TIP:Thexylenes-ethanolstepattheendoftheprocedureiscriticalforsubsequentLCM.Thelengthoftimemayneedtobeadjusteddependingonthetissuetypeandgoalsofthestudy.Forexample,ifthetissueisleftinthissolutionlongerthan10-15sec,thetissuemaydetachfromtheslideduringdissection.Conversely,ifthexylenes-ethanolstepistooshort,thetissuemaybestronglyboundtotheslideandnotdissectwell. TIP:ThetissuesectionshouldbecompletelydrybeforeLCM.UseofanAccudusterorsimilardevicemayfacilitatedryingforefficientmicrodissection.