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HandEPartTwo:Method,TipsandTroubleshooting
December9,2014byPB

ThecompletemethodforH&Estainingiscontainedinthetablesbelow,butwelltakealookateachofthestagesinturn
andexplaintheprocesswhichshouldhelpyoushouldyouneedtotroubleshoot.

Dewaxandrehydrate

Thefirststageinanyhistologicalstainingorimmunohistochemistrymethodistodewaxyoursectionsontheslides.The
paraffinneedstoberemovedtoenabletheaqueoussolutionstopenetratethefixedtissue.Thisisusuallydoneusingeither
xyleneorHistoclear.Afterthewaxisremoved(typicallyinthreeseparate5minuteimmersionsindewaxingsolution),the
tissueneedstoberehydratedwhichisdonethroughadecreasingseriesofalcoholsolutions.InthesefirststagesofanH&E
protocol(oranyimmunohistochemicalmethod)theresnotmuchtotroubleshootyoushouldensurethatmostofthexylene Recentposts
isdrainedfromtherackandslidesbeforeplacingtheminthe100%alcoholandalsoensurethattheslidesareexposedto
theairfortheminimumamountoftimebetweensolutions.Forthetapwaterstagesofthisprotocol,Iwouldrecommend ReadingforPleasure,Writing
usingacontainerwithinthesinkwithconstantlyrunningwaterthishelpstocontaintheexcessstainsfromtheslideswhich forScience
wouldotherwisestainthewholesinkblueandpink!
AriseSiRHoechst!ANewFar
RedDNAStain
Santaology:TheScienceof
Gottheblues
ChristmasPresentDelivery
Asmentionedinthepreviousarticle,haematoxylinisusedtodyethenucleusandbindstothehistoneswiththeaidofthe AllSyntheticLifeisHere:The
metalsalt,otherwiseknownasthemordant.Afiveminutesubmersioninhaematoxylinisstandardthisrelativelylongtime WorkofJ.CraigVenter
ensuresthatalloftheavailablenuclearbindingsitesaresaturated.
MicroscopeCondensersThe
Thismethodofstainingisknownasregressivestaining.Thetissueisincubatedforalongperiodtoallowfordeliberate OverlookedOptics
overstainingandthensomeofthestainisremovedinastepcalleddifferentiation.Theothermethodisprogressive
stainingwherethesolutionofhaematoxylinmaynotbeasconcentratedandthetissueischeckedatregularintervalsuntil Archives
thedesiredintensityisreached.Personally,Ihavealwaysusedtheformermethodasitsprobablytheeasiestandinthe
endislesstimeconsumingasyoudontneedtokeeptakingtheslidesfromthehaematoxylin,submersingthemintap February2016
waterandcheckingtheintensityusingamicroscope. January2016

Whenyoutakeyourslidesoutofthehaematoxylin,allwillbeblue!Initially,thenucleiwillbedeeppurple/blue/,butthe December2015
differentiationstagewillrenderthestainednucleidarkblue.Ifyouoverdifferentiate,fearnot!MostofthestagesoftheH&E October2015
methodcanberepeatedandthefinalcolourcanbeadjusted.
September2015
August2015
DifferentiationandBluing July2015
June2015
Toachievethebluecolourednuclei,therearetwostages.Thefirstisthedifferentiationstage.Afterthetapwaterrinse,the
slidesaresubmergedforashortperiodoftimeinanacidalcoholsolutionwhichremovestheexcessstaining.Atthisstage, May2015
thenucleiwillbeverydarkanddeeppurpleincolour.ToachievetheclassicH&Ebluecolour,theslidesarerinsedthen April2015
submergedinasolutioncalledScottsTapWater.Thissecondstageisknownasbluing.
March2015
Theuseoftapwater(asopposedtodistilledwater)isimportantinthesestagesandbluingcanbeachievedwithtapwater February2015
alone(dependingontheintensityofthestainingfollowingthehaematoxylinstage).Tapwaterisslightlyacidic(pHof
January2015
around6.0to6.8),butthisismorealkalinethanthepHofthehaematoxylinwhichtendstobearoundpH2.7.Twotofive
minutesinrunningtapwaterwillremovemostoftheexcessmordantgivingsharpbluenuclearstaining. December2014
October2014

September2014
Eosin
July2014
TheaimofH&Estainingistoachieveacontrastbetweenthedeepbluenucleiandred/pinkoftheothercellular June2014
components.Eosinisusedtostaincytoplasm,musclefibresandsoon.Thispinkcounterstainalsohelpstodifferentiate
May2014
betweennucleiandnonnuclearcomponentsincells.Dontbetemptedtooverstainatthisstagealwayserronthesideof
caution.Overstainingcanresultincellswhicharenotimmediatelyeasytodifferentiate.Youcanremoveacertainamount April2014
ofoverstaininrunningtapwater. March2014

SomemethodssuggestthatEosinisdilutedinethanol,butIwouldrecommendasolutioninwaterastheethanolhasthe
Newslettersignup
potentialtoremovethehaematoxylinstainingfromthepreviousstages.

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Method
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ThisisabasicmethodforH&EstaininginwhichIhavesplitintotwosections:thedehydration/rehydrationsection(for
ThisisabasicmethodforH&EstaininginwhichIhavesplitintotwosections:thedehydration/rehydrationsection(for
rehydrationfollowingtheeosin/tapwaterrinse,simplyfollowStep9toStep1beforemountingtheslides).
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Dehydration/Rehydration

Step Solution Time Notes

1 Xylene 5mins Thesestepsshouldbecarried
outinafumehoodordownflow
bench
2 Xylene 5mins
3 Xylene 5mins Drainoffexcessxylenebefore
(4)
4 Absoluteethanol 20secs
5 Absoluteethanol 20secs
6 Absoluteethanol 20secs
7 95%ethanol 20secs
8 80%ethanol 20secs
9 70%ethanol 20secs
10 Tapwater 2mins Runningtapwaterinasink

H&EStaining

Step Solution Time Notes

1 HarrisHaematoxylin 5mins Runcontrolslidesfirsttocheck
timesandstaining.
2 Tapwater 20secs Runningtapwaterinasink
3 1%Acidalcohol 5secs 5secsismaximumtimeinthis
solution
4 ScottsTapWater 2mins Untiltissuesectionsturnblue
5 Eosin 2mins
6 Tapwater 20secs Runningtapwaterinasink,
thencheckslides.

Thenucleiinthecellsshouldbedarkbluewhereasthecytoplasmwillbepink.Fibrinandmusclewillbedeeppinkand
collagenwillstainpalepink.Anyerythrocyteswillbered.

Remembertoallowanyexcessfluidtodrainbackintothestainingdishbeforeproceedingtothenextstep.

Slidesaremounteddirectlyfromthelastxylenestep.

Author:MartinWilson

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HandEPartOne:History,BackgroundandSolutions UnmaskThoseAntigens:RetrievalTechniquesto
EnhanceImmunohistochemistry

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