| ApplicationNotes | Westernblot:Recognizesadiffusebandatapproximately70-85kDaonwesternblotsofextracts(20ugtotalprotein)fromhumancaudate,putamen,andnucleusaccumbens.
  Immunohistochemistry:4%paraformaldehydefixedtissue(careshouldbetakennottoover-fixtissue);perfusionfollowedbylessthan90minutespost-fixation,thencryoprotect.Suggestedworkingdilution1:1,000-1:10,000.Ifusingonrattissue,absorbedanti-ratsecondaryantibodiesarerecommended,andtheuseofratPAPsystems,orABCdetectionwillenhancesensitivity.
  Immunocytochemistry:1:5,000to1:10,000.
  Immunoblotting(notrecommendedforuseonrat)
  Optimalworkingdilutionsandprotocolsmustbedeterminedbyenduser.
 
 
  IMMUNOHISTOCHEMISTRYPROTOCOLFORMAB369
  Thisantibodyhasbeenusedsuccessfullyon30μm,freefloating,4%paraformaldehydefixedmousebraintissue.Allstepsareperformedunderconstantagitation.Suggestedprotocolfollows.
  1)3x10minutewashesinTBS(withorwithout0.25%Triton).
  2)Incubatefor30minutesinTBSwith3%serum(sameashostfromsecondaryantibody).
  3)IncubateprimaryantibodydilutedappropriatelyinTBSwith1%serum(sameashostfromsecondaryantibody)(withorwithout0.25%Triton)for2hoursatroomtemperaturefollowedby16hoursat4°C.
  4)3x10minutewashesinTBS.
  5)IncubatewithsecondaryantibodydilutedappropriatelyinTBSwith1%serum(sameashostfromsecondaryantibody).
  6)3x10minutewashesinTBS.
  7)ABCElite(1:200VectorLabs)inTBS.
  8)2x10minutewashesinTBS.
  9)1x10minutewashinphosphatebuffer(nosaline).
  10)DABreactionwith0.06%NiCladdedforintensification.
  11)2x10minutewashesinPBS.
  12)1x10minutewashinphosphatebuffer(nosaline). |