JournalofVirologicalMethods99(2002)23–32ImproveddetectionofsimianimmunodeficiencyvirusRNAbyinsituhybridizationinfixedtissuesections:combinedeffectsoftemperaturesfortissuefixationandprobehybridizationBethA.Fallert,ToddA.Reinhart*DepartmentofInfectiousDiseasesandMicrobiology,GraduateSchoolofPublicHealth,Uni�ersityofPittsburgh,Pittsburgh,PA15261,USAReceived21May2001;receivedinrevisedform26July2001;accepted27July2001AbstractInsituhybridizationdetectionofviralRNAsinformaldehyde-fixedtissuespecimensisusedfrequentlytocharacterizetheextentofviralreplicationwithinhosttissues.TheabilitytodeterminethelevelofexpressionofviralRNAsinsituisdependentuponmanyfactorsincludingtheextentofcross-linkingduringfixation,thepretreatmentregimenutilizedtorelievetheeffectsofcross-linking,andthehybridizationandwashprotocols.Ineffortstoimproveourabilitytodetectcellsinfectedproductivelybysimianimmunodeficiencyvirus(SIV)inrhesusmacaquetissues,theeffectsofunconventionallyhigh(40°C)andmorestandardlow(4°C)temperaturefixationin4%paraformalde-hyde/phosphatebufferedsalineweretestedempiricallyoninsituhybridizationsignals.Inaddition,hybridizationtemperaturesrangingbetween37and75°CwereutilizedtodeterminetheoptimalhybridizationconditionsfordetectionofSIVproductivelyinfectedcells.Fixationconditionsof40°Candhybridizationconditionsof50–55°CwereidentifiedasprovidingthegreatestsensitivityfordetectingRNA+cellsandforquantitatingthesignalpercell,whilestillallowingantigenicepitopestobedetectedbyimmunohistochemicalstaining.ThesedataindicatethatthesignalintensityfollowinginsituhybridizationforviralRNAsisdependentuponthecombinedeffectsoftissuefixationandinsituhybridizationtemperatures.©2002ElsevierScienceB.V.Allrightsreserved.Keywords:Simianimmunodeficiencyvirus;Insituhybridization;Immunohistochemistry;Formalinfixation;Temperaturewww.elsevier.com/locate/jviromet1.IntroductionInsituhybridizationisanextremelyversatileandusefulmethodfordeterminingthenumbersandlocationsofindividualcellsharboringaspe-cificnucleic...
