| ApplicationNotes | Immunohistochemistry:1:500-1:2,500usingfreefloatingsectionsbythePAPtechniqueonratdopaminergicareas.
  Optimalworkingdilutionsmustbedeterminedbyenduser.
  PROTOCOLforDopamineDetectionbyImmunohisto/cytochemistry.Exampleforaratbrain.
  1.SOLUTIONSTOBEPREPARED-Solutionmustbepreparedasneeded.
  SolutionA:0.1Mcacodylate,10g/LsodiummetABIsulfite,pH6.2.
  SolutionB:0.1Mcacodylate,10g/Lsodiummetabisulfite,3-5%glutaraldehyde,pH7.5.
  2.RATPERFUSION-TheratisanaesthetizedwithsodiumpentobarbitalorNembutalandperfusedintracardiallythroughtheaortausingapumpwithSolutionA(30mL):150-300mL/min,SolutionB(500mL):150-300mL/min.
  3.POSTFIXATION:15to30minutesinSolutionB,then4softwashesin0.05MTriswith8.5g/Lsodiummetabisulfite,pH7.5(SolutionC).
  4.TISSUESECTIONING:Vibratomeorcryostatsectionscanbeused.
  5.REDUCTIONSTEP:SectionsarereducedwithSolutionCcontaining0.1Msodiumborohydridefor10minutes.Thesectionsarewashed4timesinsolutionCwithoutsodiumborohydride.
  6.APPLICATIONOFDOPAMINEANTIBODY:Useafinaldilutionof1:2,500-1:10,000inSolutionCcontaining0.1%TritonX100and2%non-specificserum.Incubate12sectionsper2mLdilutedantibodyovernight,+4°C.Thenwashthesectionsthreetimesfor10minuteseachinSolutionC.(Notethattheantibodymaybeusableatahigherdilution.Thisshouldbeexploredtominimizethepossibilityofhighbackground.Additionally,notethatachangeinthebufferingsystemasindicatedintheprotocolmaychangethebackgroundandantibodyrecognition).ThespecificreactionisthenrevealedbyPAPprocedure.
  6.SECONDANTIBODY:Incubatethesectionswitha1:50to1:200dilutionofgoatanti-rabbitinSolutionBcontaining1%non-specificserumforeither3hrsat20°Cor2hrat37°C.Thenwashthesections,3times,for10minuteseachwithSolutionB.
  7.PAP:Incubatethesectionswiththeappropriatedilutionofperoxidaseanti-peroxidase(forfreefloatingmethod)inSolutionBcontaining1%non-specificserumfor1-2hoursat37°C.Thenwashsections3timesfor10mineachinsolutionB.
  8.VISUALIZATION:Theantigen-antibodycomplexesarevisualizedusingDAB-4-HCl(25mg/100mL)(orotherchromogen)in0.05MTrisandfiltrated;0.05%hydrogenperoxideisadded.Incubatethesectionsfor10minutesatroomtemp.Stopthereactionbytransferringthesectionsto5mL0.05MTris.WashtissuewithsolutionDusing2,10minwashes.Mountsectionsonchrome-alumcoatedslides.Dryovernightat37°C.Rehydratesectionsusingconventionalhistologicalprocedures.Coverslipusingrapidmountingmedia.
  Forresearchuseonly;notforuseasadiagnostic. |