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SilverStainingforNucleolarOrganizerRegions
TrudeSchwarzacherandMolecularCytogeneticsResearchGroup
Solutions:
Boratebuffer:0.01MNa2B4O7,pH=9.22
Aqueoussilvernitratesolution:
1gAgN03(reasonablyhighgrade,clean,andnottooold)
1mlgooddistilledwater
10ulformalin(=.37%formaldehyde,optional,increasesstaininginoldslides)
Mixwellandputinaplasticsyringewithamilliporefilter.
Donotbringincontactwithmetalobjects.
Caution:
Silvernitratestainsclotheslabbenchesandfingersblack,normallyafterabout1016h.Itiscorrosive,butnot
reallydangeroustohumanbeings,andisusedoccasionallyasadisinfectant.Theskinshedsandtheblackspots
disappearafterafewdays.Itisadvisabletowearglovesastheblackspotsarenotveryattractive.Clothsandlab
benchesarenormallysoiledforlife.
Method:
1.Incubateslidesinboratebufferfor1020min(towashslidesfromremainingaceticacid,andtoadjustfor
ahighpHwhichisfavourableforsilverstaining).
2.Rinseindistilledwater.
3.Airdry.
4.Apply200300/ulfreshlymadesilvernitratesolutiondirectlyfromthemilliporefilterontheslide
5.Coverwitha25x40mmcleannylonmesh(200300umporesize,weuseSwissSilkBoltingClothFabrik,
Zurich,Switzerland,Nybolt68GG243butIthinkothersdoaswell).Makesurenylonmeshissoaked
thoroughly.Ifnecessaryapplyanotherdropofsilvernitratesolutionontop.
6.Incubateinamoistchamberat370Cuntilgoldenbrown(about13h).Forquickresultsincubateat600C
for520min.
7.Rinseinplentyofdistilledwater.
8.Airdry.
9.Examineunderthethroughlightmicroscope.
PutafewdropsofxyleneonthepreparationandcoverwithalargeNo.0coverslip.Reapplyxyleneifitdries
out.Forstorage,leavetheslideonthebenchuntilallthexylenehasevaporated,removethecoverslipcarefully
andputslideinabox.Thismethodgivesexcellentcontrast,butyouhavetocopewithalittlebitofxylenesmell.
Alternatively,youcanexaminetheslidesdirectlywithimmersionoil.Thiswillnotdestroythesilverstaining.
Oilcanberemovedwithxylene.
Results:
Nucleishouldbevisiblewithoutphasecontrast.Theyshouldappearyellowtolightbrownandnucleolibrown.
Toolongstainingresultsinblacknucleoliandeventuallyblacknuclei.Ifblackprecipitateoccurs,slideswerenot
cleanorthesilvernitratesolutionwascontaminated.
Atmetaphase,silvernitratestainsnucleolusorganizerregionswhichhavebeenactiveinthepreceding
interphase.Althoughamountofsilvernitrateisinsomerelationtotheamountofactivity,ornumberofactive,
rDNAcopiesitcannotbeusedforanaccuratequantitativestudy,partlybecauseofdifferencesofsilverstaining
strengthswithinandbetweenslidesdependingonfixationprocedures,densityofnuclei,preparationqualityand
ageofslides.Sometimes,e.g.withrye,heterochromaticregionsalsostainapalebrowncolor.
References:
Kodamaetal.1980:Animprovedsilverstainingtechniquefornucleolusorganizerregionsbyusingnyloncloth.
JpnJHumanGenet25,229233
SchwarzacherT,KraemerPMandCramLS1988:SpontaneousinvitroneoplasticevolutionofculturesChinese
hamstercells.Nucleolusorganizingregionactivity.CancerGenetCytogenet35,119128.
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