ApplicationNotes | EvaluatedbyWesternBlotonmousebrainmembranelysates.
WesternBlot:1:500dilutionofthisantibodydetectedGLT-1on10ugofmousebrainmembranelysates.
Immunohistochemistry: 1:1,000-1:4,000dilutionfromapreviuoslotusedaDABdetectionsystemonadultratforebrainfixedwith4%paraformaldehyde.
Immunohistochemistry: 1:5,000-10,000dilutionfromapreviuoslotusedacyanineconjugatedsecondaryantibody.
Enzymaticdetectionrequiressubstantiallyhigherprimaryantibodydilutions.Lightlyfixed4%PFAmaterialisrecommended.
Notes:
MaleSprague-Dawleyrats(b.wt.100-150g)wereanesthetizedwithsodiumpentobarbitalandperfusedviatheascendingaortawith50mLofCa2+-freeTyrode+ssolutionfollowedbyaformalin-picricacidfixative(4%paraformaldehydewith0.4%picricacidin0.16Mphosphatebuffer,pH6.9)for6minutes.Tissueswererapidlydissectedout,postfixedinthesamefixativefor90minutesandrinsedforatleast24hoursin0.1Mphosphatebuffer(pH7.4)containing10%sucrose.Sectionswerecut(14um)inacryostatandincubatedat4°CovernightwithAB1783(1:5,000-1:10,000).AfterrinsinginPBSsectionswereincubatedfor60minutesatroomtemperaturewithCy3-conjugatedsecondaryantibodies.AftermountinginamixtureofPBSandglycerol(1:3)containing0.1%p-phenylenediamine,sectionswereexaminedwithaNikonMicrophot-SAepifluorescencemicroscope.
Optimalworkingdilutionsmustbedeterminedbytheenduser. |