byVincentR.Klump,Jr.,HT(ASCP),HistologyServices,EastHaven,CT

note:Mr.Klumpwasinstrumentalinthedevelopmentofastandardized,consistent,reproducIBLeprotocolformicrowavehistoprocessingintheoriginalEnergyBeamSciencesH2500MicrowaveProcessor.

MicrowaveTissueProcessing

Tissuecriteria:Maximumtissuesizeforthisprocedure:3mmx3mmx3mm.Variationsofthissizecanalsobeaccommodated,buttotaltissuevolumeshouldnotexceed3mmcubed.

Examples:3mmpunchbiopsies,coreneedlebiopsies,corebonemarrowbiopsies,shavebiopsies.

Tissueshouldbefixedin10%NBFormalinforaminimumoffourhours.

Equipment:

• Laboratorymicrowaveequippedwithatemperatureprobeaccurateto+/-1ºC
• Microwave-safecontainerholdingaminimumof250ml
• (optional)microwave-safecassetteracksuppliedbyEnergyBeamSciences
• 100%ethanol
• 100%isopropanol
• liquidparaffin
• boilingchips(preferably,commonstonesnotbiggerthan1cmx.5cmx.5cm)

Procedure:

1. Rinsetissuefor5minutesinrunningwater
2. dehydratein100%ethanolfor15minutesat65ºCinmicrowave
3. clearin100%isopropanolfor10minutesat74ºCinmicrowave
4. infiltrate(3steps)inliquidparaffinfor5minutesat65ºCinmicrowavewithboilingchips,5minutesat74ºinmicrowavewithboilingchips;freshchangeofliquidparaffinfor5minutesat82ºinmicrowavewithboilingchips
5. transfercassettestocleanparaffinandembedasusual

AntigenRetrievalusingCitrateBuffer

Slidecriteria:Positively(+)chargedorsilanetreatedprecleanedglassslides.Tissueplacedcentrallyonslides.Slidesbakedforaminimumof1hourat60ºC(preferably,bakedovernightat60ºC)

Examples:

Antigenretrievalmaybeutilizedforalmostallantibodies,enhancingstainingandallowingforgreaterantibodydilutionaswellasshorterincubationtimes.Mostantibodieswhichrequireenzymetreatmentstainremarkablywellutilizingantigenretrievalprocedures.Remembertoalwaysrunaknownpositivecontrolwitheachantibodyandatruenegativewitheachcase.

Equipment:

• Laboratorymicrowavewithtemperaturecontrolaccurateto+/-1ºC
• Microwave-safestainingdish
• Microwave-safesliderack
• Distilledwater
• CitricAcid
• SodiumHydroxide
• pHmeter

Procedure:

1. Xylene,2changes,10minuteseach
2. 100%ethanol,twochanges,1minuteeach
3. 2%HydrogenPeroxideMethanolquenchfor10minutes
4. 100%ethanol,twochanges,1minuteeach
5. 95%ethanol,twochanges,1minuteeach
6. 70%ethanol,1minute
7. Rinseindistilledwater,1minute

Citratebuffer:10mMCitrateBuffer(2.1gCitricAcidto1Lofdistilledwater,adjustpHto6.0withapproximately10mLof2MNaOH).Immersesectionsincitratebufferfor10minutesat100ºCinmicrowave.

Note:Slides*must*cooltoroomtemperaturebeforeproceedingtoIHCsteps.Rinseslidesindistilledwater,transfertobuffer,andgoontoIHCprocedure.Avarietyofstainingtechniquesaresuitedtothisprocedure,includingAPAAP,PAPandABCComplex/HRP.

Note:ThisprocedurewasadaptedfromDakoAntibodyDataSheetforKI-1

SpecialStainsUtilizingtheMicrowavePAS,PAS+DIASTASE,Fe++

Weareallveryfamiliarwithsilverstainsinthemicrowave,butthesethreestainsarehistochemicalstainswhichthemicrowaveacceleratesconsiderably.

PAS+DIASTASEProcedure:

PAS+DIASTASEdoneatroomtemperaturetakestwohours.PAS+DIASTASEaidedbythemicrowavetakes50minutes.

Equipment:

• Laboratorymicrowavewithtemperaturecontrolaccurateto+/-1ºC
• Microwave-safeCoplinjars
• .05%PeriodicAcid
• Schiff"sReagent
• Hematoxylin,GillIII
• Diastaseofmalt,reagentgrade,.5gram/50mldistilledwater(mixwell,makefresh)

Procedure:

1. Bake,deparaffinizeandhydrateslidesaccordingtostandardprotocol
2. Microwaveslidesfor15minutesat37ºCindiastasesolution
3. Rinsefor10minutesinrunningwater
4. .05%PeriodicAcidfor5minutes
5. Rinsefor1minuteinrunningwater
6. Microwaveslidesfor1minuteat30ºCinSchiff"ssolution(besuretoeithermixsolutionoruseairbubbleagitatortoequalizetemperature).
7. Leaveslidesfor5minutesinthissolution.Note:Thisstep*must*bedoneunderahood.
8. Washintapwateratroomtemperatureuntilapinkcolordevelops(atleast1minute).
9. CounterstaininGillIIIHematoxylinfor1minute
10. Bluerinseintapwater;dehydrate,clearandmount

Note:Thisprocedurewasadaptedfrom"TheMicrowaveCookbookforMicroscopists",BoonandKok,CoulombPressLeiden,1992

PASProcedure:

PASdoneatroomtemperaturetakes45minutes.PASaidedbythemicrowavetakes15minutes.

Equipment:

• Laboratorymicrowavewithtemperaturecontrolaccurateto+/-1ºC
• Microwave-safeCoplinjars
• .05%PeriodicAcid
• Schiff"sReagent
• Hematoxylin,GillIII

Procedure:

1. Bake,deparaffinizeandhydrateslidesaccordingtostandardprotocol.05%PeriodicAcidfor5minutes
2. Rinsefor1minuteinrunningwater
3. Microwaveslidesfor1minuteat30ºCinSchiff"ssolution(besuretoeithermixsolutionoruseairbubbleagitatortoequalizetemperature).
4. Leaveslidesfor5minutesinthissolution.

   Note:Thisstep*must*bedoneunderahood.

5. Washintapwateratroomtemperatureuntilapinkcolordevelops(atleast1minute).
6. CounterstaininGillIIIHematoxylinfor1minute
7. Bluerinseintapwater;dehydrate,clearandmount

Note:Thisprocedurewasadaptedfrom"TheMicrowaveCookbookforMicroscopists",BoonandKok,CoulombPressLeiden,1992

Fe++Procedure:

Fe++doneatroomtemperaturetakes30minutes.Fe++aidedbythemicrowavetakeslessthan2minutes.

Equipment:

• Laboratorymicrowavewithtemperaturecontrolaccurateto+/-1ºC
• Microwave-safeCoplinjars
• 2%PotassiumFerrocyanide
• 2%HydrochloricAcid
• NuclearFastRedSolution

Procedure:

1. Bake,deparaffinizeandhydrateslidesaccordingtostandardprotocol
2. Prepareworkingsolution:
3. 25mlof2%PotassiumFerrocyanide,25mlof2%HydrochloricAcid(preparefreshbeforeuse)
4. Placeslidesinmicrowave-safeCoplinjarwith50mlofworkingsolution
5. Microwavefor40secondsathighpower(donotusetemperatureprobeinthisstep,asthereisapotentialforreactionbetweenmetalprobeandworkingsolution)

Note:Allmicrowavesvaryinpoweroutput;adjustmicrowavepowerleveltoacheivefinaltemperatureof60ºC.Rinsewellindistilledwater.CounterstaininNuclearFastRedfor15secondsat60ºCusingtemperatureprobeRinseintapwater,dehydrate,clearandmount

Note:Thisprocedurewasadaptedfrom"TheMicrowaveCookbookforMicroscopists",BoonandKok,CoulombPressLeiden,1992

MicrowaveStimulatedDecalcification

Examples:

BonebiopsiesareusuallydecalcifiedforaperiodrangingfromonehourtodaysinaweakhydrochloricacidsolutionorinEDTA.Withtheaidofmicrowaveexposure,thetimeisacceleratedfromonehourusingconventionalmethodstoaslittleas10minutesforbonemarrowcorebiopsiesusingformicacid.

Equipment:

• Laboratorymicrowaveequippedwithatemperatureprobeaccurateto+/-1ºC
• Microwave-safecontainerholdingaminimumof250ml
• (Optional)MicrowavecassetteracksuppliedbyEnergyBeamSciences
• 5%FormicAcidSolution

Procedure:

1. Keepbonebiopsiesasthinaspossible
2. Fixforstandardtimesin10%NBFormalin
3. Placethebonebiopsiesinamicrowave-safecontainerwithatleast10xvolumeof5%FormicAcidSolutiontovolumeoftissue
4. Microwavefor10minutesat55ºC
5. Repeatthisprocedureuntildesiredsoftnessisachieved
6. Rinseinrunningtapwaterforatleast10minutes
7. Proceedwitheitherconventionalormicrowaveprocessing

Note:

Treatmenttimedependsonthicknessandondensity:solidboneandteethtakemoretimethanspongybone.Bonemarrowbiopsiesrequireaslittleas10minutesexposuretime.

MicrowaveStimulatedFixation:AdvantagesandDisadvantages

Examples:

Standardfixationprotocolsinformalintakeaminimumoffourhoursatroomtemperature.Fixationaidedbymicrowaveexposureandpre-andpost-treatmentinformalintakelessthan30minutes.

Equipment:

• Laboratorymicrowaveequippedwithatemperatureprobeaccurateto+/-1ºC
• Microwave-safecontainerholdingaminimumof500ml
• (Optional)MicrowavecassetteracksuppliedbyEnergyBeamSciences
• PBS0.1MpH7.4
• 10%NBFormalin
• 70%Ethanol

Procedure:

1. Placetissuein10%NBFormalinasatransportorcollectionmedium
2. Grossspecimensnolargerthan3mminthickness
3. Placeinplasticcassettes
4. Collectspecimensingroupsof24forfixation
5. Immersespecimensinapproximately500mlPBS0.1MpH7.4(cassetterackmaybeused,orgroupsof20specimensincassettesinasuitablemicrowave-safecontainer)
6. Microwaveat68ºCfor5minutes
7. Placetissuein70%Ethanol
8. Continuewithconventionalhistoprocessingormicrowavehistoprocessing

Advantages:

10%NBFormalincanvirtuallybeeliminatedfromthelaboratory.Immunostainingisgreatlyenhancedinmicrowave-fixedtissue.Timeissavedversusconventional4hourfixation.

Disadvantages:

Manylaboratoriesaregenerallyresistanttochangesinestablishedprocedures.Nocontroloftimeinformalinwhenspecimensarereceivedfromoutsidethelab.Immunostainingresultsmayvaryduetochangingtimesinformalinandincreasedcross-linking.

Conclusion:

Manyfactorsmustbeconsideredwhenthinkingaboutswitchingtomicrowavefixationoftissueratherthanconventionalfixation.First,themannerinwhichspecimensaretransportedtoyourlaboratoryisthemostcriticalfactorincontrollingpre-fixationtimeinformalin.Anyreferencelaboratoryorlaboratoryreceivingspecimensthroughthemailgenerallyreceivethesespecimensalreadyfixed.Theselaboratoriescanconsiderremovingformalinfromthetissueprocessorandstoringspecimensin70%Ethanolforamorechemically-friendlylaboratory.

Thelaboratorieswhichprocesssamplesbiopsiedattheirownmedicalcenterorpickedupbyalocalcouriercanbestutilizemicrowavefixation.Theselabscancontroltimesinformalinpre-fixation,whichcanleadtomoreconsistentimmunoresults.

Anotherfactortobeconsideredisthechangeinschedulinginthestandardlaboratoryroutine.Schedulingchangeswillaffecteveryone,fromthecouriertotheclericalpeopleinvolvedinreporting.

Iconcludethatmicrowavefixationisnotadaptabletoeverylaboratory,but,inthosesituationswherespecimentransportcanberegulated,itcanbebothcost-effectiveandmorehealthyfortechniciansandtheenvironment.Keepinmindthatanylaboratorycanutilizethistime-savingprocedureforstatbiopsies,whichcanultimatelycontributetobetterpatientcareandagoodoptionforfastspecimenturn-around.

SlideDryingintheMicrowaveExample:

Slidedryinginaconventionalovenisdoneattemperaturesbetween60ºCand80ºC,fortimesrangingfrom20to60minutes.Slidedryinginamicrowavetakesfrom2to5minutes.

Equipment:Laboratorymicrowavewithoutputpowerfrom600-900wattsandequippedwithacarousel.Microwave-safeslideracksholding20-24slides

Procedure:

• Cutstandardparaffinsectionsfrom4-6umandplaceonglassmicroscopeslides
• Placeslidesinmicrowave-safesliderack
• Drainwateroffofslidesbygentlytappingoverpapertowelorblotter
• Placeslidescentrallyonmicrowavecarouselandmicrowaveathighpowerfor2-5minutes

  Note:Timeswillvarywithoutputpowerofmicrowave,andmustbedeterminedexperimentallyineachlaboratorywithitsmicrowave

• Letslidesbrieflycoolandcontinuewithstandardstainingprocedure

Note:Whenconsequetivebatchesaredriedinamicrowave,themicrowavecavitytemperaturewillgetgraduallyhigher.Whennotdryingslides,keepcavitydooropentoallowforcoolingbetweenbatches.

MicrowaveTissueProcessingQCChart

**Optionalchangeofparaffin

*ThismicrowaveprocessorhasbeenreplacedbytheH2800