Bejerano Lab

http://bejerano.stanford.edu
Asof3/2/11

ParaffinEmbeddingProtocol
Day1
Materials:
1XPBS
Ethanol(30%,50%,60%,70%,dilutedwithddH2O)
Glassvialswithscrewonlids
Orbitalrocker

Procedure:
1.)Ifembryosareinsucrose,doseveralwashesbackinto1XPBS.

3washesx30

2.)Dehydratetissueusingthefollowingwashes:

30%EtOH

1hour

RT

Rocking

50%EtOH

1hour

RT

Rocking

50%EtOH

1hour

RT

Rocking

60%EtOH

1hour

RT

Rocking

70%EtOH

overnight

RT

Rocking

Notes:
Shoulddolonger1XPBSwashesiftimeallows.
Useatleast200proofethanol.
Doethanolwashesquickly.Donotleaveembryosexposedtoair.

Bejerano Lab

http://bejerano.stanford.edu
Asof3/2/11

ParaffinEmbeddingProtocol
Days24
Materials:
Ethanol(85%,95%,dilutedwithddH2O,100%,)
Citrisolv/CitrisolvHybrid
Orbitalrocker
Paraffin
WarmParaffinbeadtray

Procedure:
1.)Useembryosfromtheovernightwashin70%ethanolmadeonDay1.Dothefollowingsteps:

85%ethanol

1hour

RT

Rocking

95%ethanol

1hour

RT

Rocking

100%ethanol

30

RT

Rocking

100%ethanol

1hour

RT

Rocking

1:1Citrisolv:EtOH

1hour

RT

Rocking

100%Citrisolv

30

RT

Rocking

100%Citrisolv

1hour

RT

Rocking

1:1Citrisolv:Paraffin

1hour

60oC65oC

inbeadtrays

100%Paraffin

1hour

60oC65oC

inbeadtrays

100%Paraffin

3nights

60oC65oC

inbeadtrays

Notes:
CanuseHistoclearorXyleneinsteadofCitrisolvifneeded.

Bejerano Lab

http://bejerano.stanford.edu

Asof3/2/11

ParaffinEmbeddingProtocol
Day5
Materials:
Vacuumoven
Metalheatblock(fortransportofvials)
Paraffinmoldsandembeddingrings
Microscope(fitsontotopofparaffindispenser)
Sharptippedtweezers
Smallplasticlid(forcontainingworkingembryosonparaffindispenser)
Benchpad(toprotectclothes)

Procedure:
1.)Turnvacuumovenonatleast1hr.beforeneeded.Makesureheatblockfortransportofvialisin
theoven.

2.)Takevialoutofbeadcontainerinparaffindispenser.Pouroutparaffininwastecontainer.Quicklyfill
containerwithfreshparaffin.Ifdoingmorethanonevial,putvialsinbeadcontainersuntilreadyfortransport.

3.)Putvialinheatblockfortransporttooven.Loosenlid.
4.)Putvialintooven(leaveinheatblock).Closedoorandlatchcompletely.
5.)Tightenvent(blackknobontoprightsideofoven).Turnvacuumon(makesurevacuumportisopen
bylooseningblackknobontopleftsideofoven).Waituntilpressurereaches15Hg,thentightenvacuumportknob
tocloseitoff.

6.)Leavevialinovenfor1hr.

Duringthisstep,labelparaffinembeddingringsandsetupinroomwiththeparaffindispenser.

7.)Removeheatblockandvialfromovenandtransfervialstobeadtraysinparaffindispenser.
8.)Pourembryoandparaffinoutintosmalllidrestingonheatedsurfaceofparaffindispenser.
3

Bejerano Lab

http://bejerano.stanford.edu

9.)Fillclearparaffinmoldwithparaffin.Restonheatedsurface.
10.)Arrangeembryoincorrectorientationinwarmparaffin(Usemicroscopeifneeded).
11.)Movemoldtounheatedsurfaceandletparaffinhardenslightlywhilemaintainingcorrectposition
ofembryo.

12.)Addalittlebitofwarmparaffintotopofclearmold.Swirlaround,thenquicklypressembedding
ringontotheclearmold.Filltherestofthemoldtothetopoftheembeddingring.

13.)Lethardenonbenchtop.SampleswillkeepinthemoldatRTuntilreadytodosectioning.

Bejerano Lab

http://bejerano.stanford.edu

ParaffinSectioningProtocol
Day6Sectioning
Materials:
Microtome
Tweezers
Paintbrush
Citrisolv
Warmdryingplatform
Woodenprobe

Warmwatertray
MetalProbe
Glassslides(SuperfrostPlus)
Slidedryingrack
Embeddedsample

Procedure:
1.)Heatwaterinwatertray.Turnlightonwhileusingthewatertray.

Letwaterheatforatleast1hourbeforeneeded.

2.)CleanbladeandaroundcuttingsurfacewithakimwipeandalittlebitofCitrisolv.Letdrycompletely.
3.)Cutoffexcesswaxfromembeddedsample.

Cutoffexcesswaxfromembeddingring.Canalsotrimaroundsampleifdesired.

4.)Lockmicrotomebladeinplaceandmakesurebladeguardsareclosed.Lockmicrotomehandleandclampembedded
sampleontomicrotome.
5.)Adjustsamplepositionsoitsitsstraightandevenrightabovetheblade.Lockintoplace.
6.)Unlockandquicklyturnhandleuntilsamplestartscuttingalittle.Trytocapturethefirstfullsectionusingwooden
probetoencouragefuturesectionstosticktoeachotherandcreatearibbonofparaffinsections.

Use510micronthicknessforsections.
7.)Gentlyplacecutsectionsintowarmwatertrayusingtweezersandmetalprobe.
8.)Usemetalprobetogentlymaneuverthesectionsontoaglassslide.
9.)Letslidedryuprightindryingrackuntilmostofthemoistureisgone.
10.)Transferslidestowarmdryingplatformtofinishdrying.Leaveonplatformforatleast1hour.
11.)Storeinplasticslidecarrieruntilreadytostain.
5

Bejerano Lab

http://bejerano.stanford.edu

ParaffinStainingProtocol
Day7Staining
Materials:
100%Citrisolv(3stainingdishes)
100%ethanol,200proof(2stainingdishes)
90%ethanol(1stainingdish)
70%ethanol(1stainingdish)
50%ethanol(1stainingdish)
ddH2O(1stainingdish)

NuclearFastRed(NFR)(1stainingdish)
Sliderack

Procedure:
1.)Loadsliderackwithslides.
2.)Dothefollowingseriesofdewaxing,dehydrating,staining,andcleaningsteps:
Reagent
100%Citrisolv
100%Citrisolv
100%Citrisolv
100%ethanol
100%ethanol
90%ethanol
70%ethanol
50%ethanol
ddH2O
ddH2O
ddH2O
DilutedNFR
ddH2O
ddH2O

SoakTime(minutes)
10
10
10
5
5
3
3
3
3
3
3
1veryquickdip
5
5

EmptystainingdishaftereachddH2OwashandrefillwithfreshddH2O.
PourNFRintostainingdishrightbeforeusing.Pourbackintoopaquecontainerimmediatelyafterstainingslides.
3.)Gentlyblotslidesinsliderackonpapertowelstoremoveexcesswater.
6

Bejerano Lab

http://bejerano.stanford.edu

4.)Removeslidesfromrackandlayonpapertowel.Letdryforrestofdayorovernight.

Bejerano Lab

http://bejerano.stanford.edu
Asof10/17/11

ParaffinStainingProtocol
Day8AddingCoverslip
Materials:
Aquamount
24x50mmcoverslips
22x50mmcoverslips
22x60mmcoverslips
Kimwipes

Procedure:
1.)Makesureglassslideiscompletelydry.Onapapertowel,laydowncoverslip,dotcoverslipwithAquamount,slowly
putglassslide(sectionsidedown)ontopofcoverslip,andgentlypushdownonslidetosqueezeoutbubblesandexcess
Aquamount.

AnothermethodistoputAquamountinsmalldotsdirectlyonslide(sectionsideup),thenslowlyaddcoverslip
andlightlypressesaroundsections.

2.)Letslidesdry~1hr.

3.)VisualizewhileAquamountisstillslightlywetforbestresults.

YoucancleanexcessAquamountoffofslidesusingwaterandkimwipesifneeded.
Aquamounttendstoshrinkexcessivelywhendry.