SlidePreparationforManualMicrodissection(forSubsequentDNA,RNA,andProteinAnalysis)

Thesemethodsweresuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy.

Manualmicrodissectionandsubsequentmolecularanalysiscanbecarriedoutonslidesstainedusingstandardhematoxylinandeosinmethods.However,ifcelltypesthatare(orarenot)expressingaspecificproteinarerequiredforastudy,thenmoreadvancedslidepreparationmethodssuchasImmuno-LCMmaybeutilized.

1.Materials

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

   Important:Forallproteinanalysis,dissolve1proteaseinhibitorcocktailtabletper10mlofeachreagent,exceptxylene.

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

• Forparaffin-embeddedsections,startatstep1.
• Forfrozen-embeddedsections,startatStep4.

Placethesectionsinthefollowingsolutions:

1. Freshxylenes(todepariffinizethesections)-5min
2. Freshxylenes-5min
3. 100%ethanol-30sec
4. 95%ethanol-30sec
5. 70%ethanol-30sec
6. Deionizedwater-30sec
7. Mayer"sHematoxylin-30sec
8. Deionizedwater-rinse15sec(x2)
9. 70%ethanol-30sec
10. EosinY-15sec
11. Deionizedwater-30sec(x2)
12. 3%glycerolindeionizedwater-30sec

    TIP:Soakingin3%glycerolisparticularlyhelpfulinpreparingthetissueformicrodissectionbecauseitrendersthetissuelessbrittleanddissectedtissuefragmentsareeasiertoprocure.

13. Afterremovingtheslidefromthe3%glycerolstep,shaketheslideintheairtoremovethelayerofglycerol/water.
14. Thenext5-10minsaretheoptimaltimeformicrodissection.Thetissueisdry,butretainsasoftconsistency.Ifthedissectiontakesmorethanafewminutes,thetissuewillbecomeincreasinglybrittleandthedissectedfragmentsmayberepelledastheneedleisbroughtinproximitytothetissue.Ifthetissuebecomesoverlydry,re-soakinthe3%glycerol/watersolutionfor1-2minutes.

B:Low-meltPolyester-embeddedSections

TIP:Proceedgentlywhenstainingsectionsembeddedinpolyesterwax.Eventhoughthesectionsareplacedonchargedslides,thetissuehasatendencytodetachfromtheslide,andthereforeshouldbemonitoredcarefullythroughoutthestainingprocedure.

Placethesectionsinthefollowingsolutions:

1. 100%ethanol-5min
2. 100%ethanol-5min
3. 95%ethanol-30sec
4. 70%ethanol-30sec
5. Deionizedwater-30sec
6. Mayer"shematoxylin-30sec
7. Deionizedwater-30sec
8. 70%ethanol-30sec
9. EosinY-15sec
10. Deionizedwater-30sec(x2)
11. 3%glycerolindeionizedwater-30sec
12. Thetissueisnowreadyformicrodissection.

TIP:Itisimportanttoensurethatthethincoatoffluidcoveringtheslideafterremovalfromtheglycerol/waterisremoved.Dissectionwiththisfluidlayerpresentresultsindiffusionoftissuefragmentswithpotentialfor"contamination"ofsamples.Additionally,dissectionofthetissueunderfluidproduceslargestripsoftissuethatarenoteasilyhomogenizedinextractionbuffers.