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ThisprotocolwasprovidedtousfromtheGraingerlabandmodifiedbyM.Khokhatoincludetheleadbrickforbettersquashing. 1.Place10Nieuwkoop&FaberStage26-34tadpolesintoadishofdeionized,distilledwater.2.Removetheyolkyventralportionofthetadpoleanddiscard.Belowisadrawingofastage30tadpolewiththe"yolkyventralportion"outlinedinred.
3.PlacealldorsalportionstogetherintheDIwaterandallowtostandfor20minutes.4.Pipettethedorsalhalves,carryingaslittlewaterwiththemaspossIBLe,intoanEppendorftubecontaining0.2mlof60%aceticacidinwater.5.Allowtostandfor5minutes.6.Pipetteallofthetissue,carryingaslittleaceticacidwithitaspossiblefromthetubeandplaceonapositivelychargedslide(ex.SuperfrostPlusfromFisher).7.Blotawayexcessaceticacidifnecessary.9.LeadbrickmodificationPlacealargecoverslipontheslide.Foldapapertowelsothatitisaboutthesizeofthecoverslipandplaceitontopofthecoverslip.Thenplacealeadbrickontopofthepapertowel/coverslipforabout5minutes.Heavycompressionisimportantforgettinggoodspreads.However,thecoverslipshouldnotslidearound.Preventingthecoverslipfromslidingiscrucialinordertogethighqualityspreads.Thisisparticularlyimportantwhenplacingthecoverslipontheslideandwhenremovingtheleadbrickandpapertowel.Afterfiveminutescarefullyremovetheleadbrickandpapertowel.10.Placeslideandcoverslipondryicefor5minutes.11.Removeslideandcoverslipfromdryiceandusearazorbladetogentlypryuptheedgeofthecoverslipfromthestillfrozenslide.12.Removecoverslip.13.Placeslideonapapertowelandstainthenuclei/chromosomesbycoveringtheslidewithHoechst33342[1microliterHoechst33342(stock:0.1mg/ml)in1mldistilledwater]for5minutes.WeargloveswhenworkingwithHoechst!14.Tiptheslideupandallowstaintorunofftheslideandontothepapertowel.15.MountbyplacingadropofPBS/glycerolonslide,addlargecoverslipandsealedgeswithclearnailpolish.16.ExamineslideforpresenceofstainedchromosomesusingUVfluorescenceusingahighpower(63Xorhigher)objective.17.Ifdoneproperly,thistechniqueshouldresultinhundreds,possiblythousands,ofspreadchromosomes.Observingcountablespreadsrequirespatience,butmostslidesyieldseveralcountablespreads.