| ApplicationNotes | Immunohistochemistry:1:2,500-1:10,000usingfreefloatingsectionsbythePAPtechniqueonrathippocampus. 
 Optimalworkingdilutionsmustbedeterminedbyenduser.
 
 PROTOCOLforGlutamateDetectionbyImmunohisto/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.APPLICATIONOFGLUTAMATEANTIBODY:Useafinaldilutionof1:2,500-1:10,000inSolutionCcontaining0.1%TritonX100and2%non-specificserum.Incubate12sectionsper2mLdilutedantibodyovernight,+2-8°C.Thenwashthesectionsthreetimesfor10minuteseachinSolutionC.(Notethattheantibodymaybeusableatahigherdilution.Thisshouldbeexploredtominimizethepossibilityofhighbackground.Additionally,notethatachangeinthebufferingsystemasindicatedintheprotocolmaychangethebackgroundandantibodyrecognition).ThespecificreactionisthenrevealedbyPAPprocedure.
 
 7.SECONDANTIBODY:Incubatethesectionswitha1:50to1:200dilutionofgoatanti-mouseinSolutionBcontaining1%non-specificserumforeither3hrsat20°Cor1-2hrat37°C.Thenwashthesections,3times,for10minuteseachwithSolutionC.
 
 8.PAP:Incubatethesectionswiththeappropriatedilutionofperoxidaseanti-peroxidase(forfreefloatingmethod)inSolutionCfor1-2hoursat37°C.Thenwashsections3timesfor10mineachinsolutionC.
 
 9.VISUALIZATION:Theantigen-antibodycomplexesarevisualizedusingDAB-4-HCl(25mg/100mL)(orotherchromogen)in0.05MTrisandfiltrated;0.05%hydrogenperoxideisadded.Incubatethesectionsfor10minutesatroomtemp.Stopthereactionbytransferringthesectionsto5mL0.05MTris.Mountsectionsonchrome-alumcoatedslides.Dryovernightat37°C.Rehydratesectionsusingconventionalhistologicalprocedures.Coverslipusingrapidmountingmedia.
 
 Forresearchuseonly;notforuseasadiagnostic.
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