ApplicationNotes | Immunohistochemistry:frozencryosections,5μmcryosectionswereair-driedandfixedincoldacetonefor10minutes.Blockedwith5%normalhorseserumandthenincubatedwith9D9for2hoursatroomtemperatureinahumidchamber.Typicaldilutionswillrangefrom1:100-1:1000dependinguponlottiter.Detectioniswithenhancedmethods(ABCorpolyHRP)andenzymaticsubstratesforbestresults.
Immunohistochemistryinparaffin:5μmthicklightlyfixedformalintissueswereprocessedthroughdecreasingethanolconcentrations,washedwithwater,thenheatedinamicrowavein10mMsodiumcitratepH6.0at780Wfor5minutes,followedby450Wfor10minutes.Thesectionswerethenincubatedinmethanolcontaining30%hydrogenperoxidefor30minutesandprocessedfurtherlikethecryosections.{Jussila,1998}.
WesternBlot:antibodyrecognizesVEGFR-3onreducedsamplesofHUVECandHDMECcells{primayhumanumbilicalveinendothelialcells&primaryhumandermalmicrovascularendothelialcells}.Thebandsseenatmolecularweight195kDaand125kDarepresenttheuncleavedandproteolyticallycleavedformsofVEGFR3.Thebandat175kDaisconsideredtobeintracellular,unglycosylatedprecursor{Bando,2004}.
Immunoprecipitation:1-5μLper300-500μLofcelllysate.UseproteinAorrabbitanti-mouseIgGcaptureforbestresults.
ELISA:9D9F9hasbeenusedasthedetectionantibodyforaVEGFR-3ELISA{Bando,2004}.
FlowCytometry:reactiveontransfectedNIH-3T3cellsexpressinghVEGFR3,Othercelllinesuntested.{Jussila,1998}.
Optimaldilutionswillneedtobedeterminedbytheenduser. |