ApplicationNotes | ImmunohistochemistryAnalysis:A1:500dilutionfromarepresentativelotdetectedGABAinratbraintissue.
ELISAAnalysis:ArepresentativelotfromanindependentlaboratorydetectedGABAinacompetitiveELISA.
ImmunohistochemistryProtocol:
Tissuesfixedwith4%paraformaldehydeand0-0.5%glutaraldehydegivesgoodresults.Glutaraldehydeisrequiredforantibodyreactivity.
1)Tissueisfixedwith4%paraformaldehyde,0-0.5%glutaraldehyde,0.5%potassiumdichromatein0.1MphosphatebufferatpH6.5.
2)Tissueispost-fixedovernight,vibratomesectionedin50mmandincubatedin0.05MTrisbuffer,pH6.5forthreehours.
3)Sectionsareincubatedfor18-24hoursinAB175dilutedinPBScontaining0.1%sodiumazide,0.2%TritonX-100and1%normalgoatserum.
4)FluoresceinconjugatedantibodyorABCsystemmaybeusedasthesecondaryreagent.
Note:Withoutcolchicinepretreatmentwell-stainedcellbodiesarevisIBLeinthecerebralcortex,cerebrallarcortex,superiorcolliculusandsomebrainstemraphe.Withcolchicinepretreatment,additionalcellbodystainingispresentintheinterpeduncularnucleusandthedorsalcolumnnuclei. |