ResearchpaperFromtheproteintothegraph:HowtoquantifyimmunohistochemistrystainingoftheskinusingdigitalimagingGeorgiosKokolakisa,LambrosPanagisc,EfstathiosStathopoulosb,ElpidaGiannikakib,AndronikiToscaa,SabineKrüger-Krasagakisa,⁎aDepartmentofDermatology,FacultyofMedicine,UniversityofCrete,GR-71110Heraklion,GreecebDepartmentofPathology,FacultyofMedicine,UniversityofCrete,Heraklion,GreececDepartmentofOphthalmology,MountSinaiSchoolofMedicine,NewYork,USAReceived29July2007;receivedinrevisedform10December2007;accepted12December2007Availableonline15January2008AbstractQuantitativeimmunohistochemistryisneededinordertoreliablyandaccuratelyassesstheexpressionofcellularproteinsintissue.Skinisadifficulttissueforautomatedimageanalysisduetoitsheterogeneouscompositionanditsarchitecture.Inthepresentstudyweusedapsoriaticskinmodeltocomparetheexpressionofp53andbcl-2beforeandaftertreatmentwithanti-tumornecrosisfactor-alphausingdigitalimageanalysis.DigitalphotomicrographswereacquiredandanalyzedwithScionimagesoftwareinordertoobtainthefractionofp53andbcl-2immunoreactivecells'areaoutofthetotalareainvestigated.StatisticalanalysiswithANOVArevealedasignificantincreaseofp53expressionandadecreaseofbcl-2expressioninall3epidermallayersduringthecourseoftherapy(pb0.001).Theresultswereinlinewiththeconventionalhistopathologicalevaluationusinganarbitraryscaletogradetheextentandintensityofthestaining.So,theestimationofvolumefractionofimmunohistochemicallylabelledcellsinskintissuecanbeperformedeasilyandrapidlyusingcommonlyavailableimageanalysissoftwareandprovidesreproducibleandunbiasednumericalestimationsoftheamountofcelllabelling.©2008ElsevierB.V.Allrightsreserved.Keywords:Quantification;Immunohistochemistry;Skin;Digitalimageanalysis1.IntroductionImmunohistochemistry(IHC)selectivelydetectsspecificproteinsintissuesectionsandhasbecomearoutinetechniquefordiagnosticpurposesandalsofortheassessmentoftheprognosisofcertaincancers(Loughlinetal.,2007).Furthermore,fortheunravellingofpathophysiologicmechanisms,I...
