ApplicationNotes | TISSUEEVALUATION:
Formalin-fixedandparaffinembeddedsections(6micronsthick)ofhumanskinandsquamouscellcarcinomaswereprepared.Cryostatsectionsoftheabovetissuesmayalsobeused.
IMMUNOPEROXIDASE
STAINING:
1.Thesectionstobestainedarehydratedwithphosphatebufferedsaline(PBS),pH7.4.
2.Theantiserumdiluted1:250-1:500(inPBS)isallowedtoreactwiththetissueat37*Cfor30minutes.
3.UsingPBS,thetissueiswashedfor15minutesatroomtemperatureandtreatedsequentiallywithgoatanti-rabbitgammaglobulin(diluted1:20inPBS)andperoxidaseanti-peroxidasecomplex(diluted1:100inPBS)for30minutesat37*C.Aftereachofthesesteps,thetissueiswashedwithPBSasbefore.
4.Thefinalstageofthereactionistreatmentofthesectionwithamixtureof3,3"-diaminbenzidineHCl(0.02%)inhydrogenperoxide(0.004%).Forpreservation,thesectionsaredehydrated,clearedandmounted.
IMMUNOFLUORESCENCE
STAINING:
1.SameasStep1above.
2.Thetissueisreactedwiththeantiserum,diluted1:20-1:40in(PBS)for30minutesat37*C.
3.AfterwashingofftheexcessreagentswithPBS,thesectionistreatedwitfluorescein-labeledgoatanti-rabbitgammaglobulinserum(diluted1:10inPBS)for30minutesat37*C.ThetissueiswashedwithPBSandaftermountingundersaline-glycerol,isviewedwithafluorescencemicroscope.
COMMENTS:
TheantiserumdescribedherehasbeenestablishedtogiveapositivestainingoftheS-100cellsusingtheaboveprotocols.Itisrecommended,however,thatthecustomerestablisheshisownoptimaldilutionstogivepositivestaining.Ingeneral,stainingcanoccuronfurtherdilutionoftheprimaryantiserumifthisisaccompaniedbyanincreaseintheincubationtime,e.g.24hours. |