ApplicationNotes | ThePH8Antibodycanbeusedtoidentifydopaminergicandserotonergicneuronsbyimmuno-histochemistryandforWesternblotanalysis,immunoprecipitationandimmuno-histochemistryofTYH,PAHandTRH.
PROTOCOLS
Immunohistochemistry
ThePH8Antibodycanbeusedfortheimmunohistochemicaldetectionofdopaminergicandserotonergicneuronsinhumanandratbrainstemtissue.
1.Tissueshouldbeformalinfixedandstoredinformalinpriortouseforaminimumoffivedays.
2.Cryoprotecttissueusing30%sucrosein0.1MTrispH7.4bufferfor24-72hours.
3.Cutusingasledgemicrotometo50μmthickness.
4.WashthetissuesamplesusingTrisbufferpriortocommencingthestainingprocedure.
5.Treatthetissuesamplesfor3x15minutesin50%alcohol.
6.Treatthetissuesamplesfor20minutesin50%alcoholand3%H2O2.
7.Treattissuefor20minuteswith10%normalhorseseruminTrisbuffer.ThisactstoblockendogeneousH2O2staining.
8.DilutethePH8AntibodyinTrisbuffer,addtothetissuesamplesandincubatefor1-3days.Recommendeddilutionsare1:2,000-1:10,000.Athighantibodyconcentrationallmonoaminergicneuronsarestainedwithnodistinctionbetweenserotonegicandcatecholominergiccells.Bydilutingtheantibodyconcentrate,celltypesaredistinguishableduetovariationinstainingintensity.Atlowerconcentrations(1:5,000-1:10,000dilution)onlyserotonergiccellswillstain.
9.Washtissue(3x15minutes),thenaddabiotinylatedanti-mousesecondaryantibodyandincubateonanorbitalshakeratroomtemperature(RT)for1hour.
10.Washtissue(3x15minutes)andincubateonanorbitalshakeratRTfor1hourwiththetertiarycomplex(ELITEKIT,Vector,USA).
11.Washtissue(3x15minutes)andincubateonanorbitalshakeratRTfor10minuteswithTrisbuffereddiamino-benzidinesubstrate.Add0.1%H2O2andincubateonanorbitalshakeratRTforafurther5minutes.
12.Mounttissueontogelatinisedslidesandallowtodrypriortomicroscopy. |