ApplicationNotes | TISSUEEVALUATION:
Formalin-fixedandparaffinembeddedsections(5-6micronsthick)ofhumansmallintestinewereusedintheevaluationofthisantiserum.
IMMUNOPEROXIDASE
STAINING:
1.Thesectionstobestainedarehydratedtophosphatebufferedsaline(PBS,pH7.4).
2.Theantiserumdiluted1:1,000-1:2,000(inPBS)isallowedtoreactwiththetissueat37*Cfor30minutes.
3.UsingPBS,thetissueiswashedfor15minutesatroomtemperatureandtreatedsequentiallywithgoatanti-rabbitgammaglobulin(diluted1:20inPBS)andperoxidaseanti-peroxidasecomplex(diluted1:100inPBS)for30minutesat37*C.Aftereachofthesesteps,thetissueiswashedwithPBSasbefore.
4.Thefinalstageofthereactionistreatmentofthesectionwithamixtureof3,3"-diamin-benzidineHCl(0.02%)inhydrogenperoxide(0.004%).Forpreservation,thesectionsaredehydrated,clearedandmounted.
IMMUNOFLUORESCENCE
STAINING:
1.SameasStep1above.
2.Thetissueisreactedwiththeantiserum,diluted1:20-1:40in(PBS)for30minutesat37*C.
3.AfterwashingofftheexcessantiserumwithPBS,thesectionistreatedwithfluoroscein-labeledgoatanti-rabbitgammaglobulinserum(diluted1:10inPBS)for30minutesat37*C.ThetissueiswashedwithPBSandaftermountingundersaline-glycerol,isviewedwithafluorescencemicroscope. |