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FREEZING TISSUE FOR CRYOSTAT SECTIONING

Alltissuetypesexceptmuscle:

  1. Tissueshouldpreferablybesnap-frozenbyquenchinginliquidnitrogen.
  2. QuenchingisaccomplishedbyputtingasmallamountofOCTcompoundontoacorkdisc,placingthefreshlybiopsiedtissueontothatandthendroppingthediscupsidedownintotheliquidnitrogen.
  3. Uponreachingthelaboratorythefrozensampleisattachedtoacryostatchuck.ToachievethisputasmallamountofOCTcompoundortragacanthgumontoachuck.Quicklyremovethesampleonthecorkdiscfromtheliquidnitrogen,placeitontopofthetragacanthgumandre-immersethewholeintheliquidnitrogenwithapairoflonghandledforcepsuntilfrozen.
  4. Thesampleisthenputintothecryostatwhereitmustbelefttowarmtocryostattemperaturebeforecutting,15-20minutesisusuallyadequate.However,urgentspecimensfromtheoperatingtheatreneedtobecutassoonaspossIBLesosectioningshouldbeattemptedimmediately.
  5. IfthesamplereachesthelabwithoutthebenefitofliquidnitrogenquenchingitmaybefrozenusingCO2apparatus.
  6. Ifthesamplehasbeenfixeditmustbewashedinrunningwaterforseveralhourstoremovethefixativeoritwillnotfreezeproperlyandcuttingwillbeverydifficult.

UseoftheCO2freezingapparatus:

  1. MakesuretheCO2cylindertapisturnedOFFandthatthetaponthefreezingapparatusisOPEN(anti-clockwise).
  2. PlaceasmallamountofOCTcompoundortragacanthgumontoachuck.Placethetissuesampleontothisandattachthechucktothefreezingapparatus.Makesurethechuckisheldfirmlybytighteningthescrews.
  3. Attachthesafetycover.
  4. TurnthetaponthefreezingapparatusclockwisetoCLOSEit.
  5. TurnONtheCO2cylindertap.
  6. HolddownthesafetylidwithonehandwhilstturningONthetaponthefreezingapparatus(anti-clockwise)toreleaseCO2gas.Useshortburstsuntilthesampleiscompletelyfrozen.
  7. CLOSEthetaponthefreezingapparatus(clockwise).
  8. TurnOFFtheCO2cylindertap.
  9. OPENthetaponthefreezingapparatus(anti-clockwise)toreleaseanyremaininggas.
  10. Removethesafetylid,releasethechuckandplaceitinthecryostat.Allowtoequilibratefor10-15minutesbeforecutting.

FROZENSECTIONSFROMMUSCLEBIOPSIES:

Muscleistheexceptionwhenfreezingtissueforcryotomy.Itmustbefrozenusingcoldiso-pentaneoricecrystalartefactwilloccur.(Thisshouldincludeothertissuecontainingmusclesuchasfull-depthgutsamples.)

  1. Takeunfixedmuscleandcuttoanappropriatesizeandorientation.
  2. PlacethetissueonaspotofOCTonacorkdiscsothatyouwillcutT/S(transversesection).
  3. PutasmallamountofliquidnitrogeninaThermosflask-about5cmdeep.
  4. Placeacold-resistantplasticbeakercontainingsomeiso-pentaneintotheliquidnitrogen.
  5. Waituntiltheiso-pentaneissyrupyandalmostsolidifying.Ifitdoessolidifytakeitoutofthenitrogenandstiritwithaplasticspatulauntilitismostlyliquidagain.
  6. Plungethecorkdiscs,tissuesidedown,intotheiso-pentane.Thiscanbedoneoutofthethermosflask.
  7. Attachthecorkdisctoacryostatchuckasdescribedabove.
  8. Putthechuckintothecryostatandleavetoreachtemperaturefor15-20minutes.

USINGTHESLEECRYOSTAT:
Temperaturegaugeandlightswitch.
Micrometerandanti-rollplatecontrol.
Maincuttinghandleandadvancemechanismhandle.
OCTcompound,corkdiscandchuck.
Internalviewofadvancemechanismandanti-rollplate.

  • Openthemainwindowandremovetheinsulatingblock.
  • Switchontheinternallight,theswitchisdownnearthetemperaturegauge.
  • Checkthatthetemperatureisat-20oCwhichisthestandardoperatingtemperature.Ifyouarecuttingsofttissuesuchasliveryoumayneedtoraisethetemperatureto-15oCandanyspecimenthathasbeenformalinfixedisprobablybestcutat-10oC.Hardertissuesproducebettersectionsatlowertemperaturesandviceversa.
  • FixedtissueMUSTbewashedinrunningtapwaterbeforefreezing.Seeabove.
  • Oncethetissueisattachedtothechuckitshouldbeleftinthecryostatforanadequatelengthoftimetoallowittoreachtheselectedoperatingtemperature.Seeabove
  • Checkthatthecryostatadvancemechanismisatthetopofitstravelonthecentralthread.Ifitisn"trotatethemaincuttinghandlesothatthechuckholderisatthelowestpointoftravelandthenwindthesmallhandleonthetopofthecryostatinaclockwisedirection.Keepdoingthisandwatchtheadvancemechanismarmtravelupthecentralthreaduntilitalmostreachesthetop,don"tgotoofaroritmaystick.
  • Attachthechucktothecryostatarmandtightenbothwingnutstoholditfirmly.Trytoalignthetissuesothatit"sflattestsideisparalleltotheknifeedge.Thiswillhelptoachieveevencuttingoftheblockandpreventsectionsfromcrumpling.
  • Iftheknifeistoofarforwardortoofarbackyoucanmovetheassemblybyreleasingthehandleontherightoftheknifeholder;pushitawayfromyou.Slideittothedesiredpositionandtightenthehandleagain.
  • Adjustthesectionthicknessusingthemicrometerscrewonthelefthandsideofthecryostat.Mosttissuescutwellat6-8µbutyoumayhavetoincreasethisfordifficulttissues.
  • Iftheanti-rollplateisinpositionrotateitoutofthewayusingthesmallhandleontheleft-handsideofthecryostat.
  • Trimthetissueblocksothatyouhaveacompletesectionthroughthetissue.Thiscanbeachievedbysimplywindingthemaincuttinghandle(takesalongtime)orbywindingthehandlewhilstalsowindingthesmallhandleanti-clockwiseontopofthecryostat.Thiscanbealittletrickyatfirstbutisthequickestwayoftrimmingablock.
  • RemovealltracesoftissueandicefromtheknifeedgebybrushingfirmlyUPWARDSwithastiffbristlepaintbrush.
  • Usethehandleontheleftofthecryostattobringtheanti-rollplatebackintoposition.Itshouldbeexactlyparalleltotheknifeedgeandwhenlookingatitthroughthesmallcircularwindowitshouldalsolineupwiththeknifeedge.failuretocutsectionsismostoftencausedbyamis-alignedanti-rollplate.
  • Turnthemaincuttinghandletoproduceasection.Thespeedatwhichyouturnthehandleandcutthesectiondependsonthetypeoftissueyouarecutting.Generallytheharderthetissuethefasteryouneedtocutit.Usuallymorethanonesectionorevenaribbonofsectionsarecutforeachslide.Thisincreasesthechancesofhavingatleastonegoodsectionperslide.
  • Topickupthesectionshaveaslideready.Useplainslidesforunfixedtissueandslidescoatedwithgelatineorpoly-l-lysineforfixedtissue.Rotatetheanti-rollplateoutoftheway.Holdtheslidebetweenyourforefingerandthumb,openthesmallwindowandplacetheslideendagainstthelefthandknifeclampandswiveltheslidedownontothesectionsandholditthereforafewsecondstoallowthesectionstomeltontotheslide.
  • Removetheslideandallowittodryatroomtemperatureforseveralminutesbeforeyouattemptanystaining.
  • Againbrushoffallremainingtissueandicefromtheknifeedgemakingsureyoubrushinanupwardsdirection.
  • Repeatasnecessary.Whenyouaredoneremovealltissuewastefromthemachineandresettheadvancemechanismtothetopofitstravelforthenextuser.
  • Replacetheinsulatingblockandswitchofftheinternallight.


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