Thesemethodsweresuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy.

LCMandsubsequentmolecularanalysiscanbecarriedoutonslidesstainedusingstandardhematoxylinandeosinmethods.However,ifcelltypesthatare(orarenot)expressingaspecificproteinarerequiredforastudythenmoreadvancedslidepreparationmethodssuchasImmuno-LCMmaybeutilized.

1.Materials

1. 70%,95%,100%ethanol
2. Xylenes,mixed,ACSreagent(Sigma)
3. Deionizedwater
4. Hematoxylinsolution,Mayer"s(Sigma)
5. EosinYsolution(Sigma)
6. Complete,miniproteaseinhibitorcocktailtablets(RocheCorp.)

Important:Forallproteinanalysis,dissolve1proteaseinhibitorcocktailtabletper10mlofeachreagentexceptthexylenes.

2.StorageofSections

• Recutparaffinsectionsarestoredatorbelowroomtemperature.Donotdeparaffinizeuntilimmediatelypriortomicrodissection.
• Low-meltpolyestersectionsarestoredat4°C.
• Frozensectionsarestoredat-80°Corbelow.

3.Methods

TIP:Usetheminimalamountofstainingtovisualizethetissueformicrodissection.Thiswillsignificantlyimprovemacromoleculerecovery.Forexample,hematoxylinandeosincanbeusedat10%oftheirstandardconcentrations.Sincetheslidesaremicrodissectedwithoutacoverslip,thetissueisnotindex-matchedandsubstantiallightscatteringoccurs,typicallyproducing"dark"images.Thus,bothimagequalityandmolecularrecoverycanbeimprovedbydecreasingstainconcentrations.A:Paraffin-embeddedSectionsorFrozenSections

• Ifaparaffin-embeddedsectionistobestained,startfromstep1.
• Ifthesectionwasfrozen-embedded,meltitgently(e.g.,onthebackofthehand)forapproximately30secafterremovalfromthefreezer.Thiswillcreatea"rougher"tissuesurfaceandallowforbetteradhesiontotheLCMcap.StartatStep4.

Placethesectionsinthefollowingsolutions:

1. Freshxylenes(todepariffinizethesections)-5min
2. Freshxylenes-5min
3. 100%ethanol-15sec
4. 95%ethanol-15sec
5. 70%ethanol-15sec
6. Deionizedwater-15sec
7. Mayer"sHematoxylin-30sec
8. Deionizedwater-rinse(x2)-15sec
9. 70%ethanol-15sec
10. EosinY-5sec
11. 95%ethanol-15sec
12. 95%ethanol-15sec
13. 100%ethanol-15sec
14. 100%ethanol-15sec
15. Xylenes(toensuredehydrationofthesection)-60sec
16. Air-dryforapproximately2minutesorgentlyuseairguntocompletelyremovexylenes.
17. ThetissueisnowreadyforLCM.

B:Low-meltPolyester-embeddedSections

TIP:Proceedgentlywhenstainingsectionsembeddedinpolyesterwax.Eventhoughthesectionsareplacedonchargedslides,thetissuehasatendencytodetachfromtheslideandshouldbemonitoredcarefullythroughoutthestainingprocedure.

Placethesectionsinthefollowingsolutions:

1. 100%ethanol(toremovepolyesterwax)-5min
2. 100%ethanol-5min
3. 95%ethanol-15sec
4. 70%ethanol-15sec
5. Deionizedwater-15sec
6. Mayer"shematoxylin-30sec
7. Deionizedwater-15sec
8. 70%ethanol-30sec
9. EosinY-5sec
10. 95%ethanol-15sec
11. 95%ethanol-15sec
12. 100%ethanol-15sec
13. 100%ethanol-15sec
14. 50:50,xylenes:100%ethanol-10sec
15. ThetissueisnowreadyforLCM.

TIP:Thexylenes-ethanolstepattheendoftheprocedureiscriticalforsubsequentLCM.Thelengthoftimemayneedtobeadjusteddependingonthetissuetypeandgoalsofthestudy.Forexample,ifthetissueisleftinthissolutionlongerthan10-15sec,thetissuemaydetachfromtheslideduringdissection.Conversely,ifthexylenes-ethanolstepistooshort,thetissuemaybestronglyboundtotheslideandnotdissectwell.

TIP:ThetissuesectionshouldbecompletelydrybeforeLCM.UseofanAccudusterorsimilardevicemayfacilitatedryingforefficientmicrodissection.