ApplicationNotes | Immunohistochemistry:1:100-1:250.Seeimmunohistochmistryprocedurebelow.
Optimalworkingdilutionsmustbedeterminedbytheenduser.
IMMUNOHISTOCHEMISTRYPROCEDURE(PAPTECHNIQUE)FORMAB305,MONOCLONALANTIBODYTOCHOLINEACETYLTRANSFERASE
I)Perfusion&SectioningProcedure
1.Perfusethroughtheheartwithafixativesolutioncontaining4%paraformaldehydein0.12Mphosphatebuffer(pH7.3)forlightmicroscopy(LM),andadditionally,0.1%gluteraldehydeand.002%CaCl2forelectronmicroscopy(EM).
2.Removebrainandpostfix2-18hoursat4°Cin4%paraformaldehydein0.12Mphosphatebuffer.
3.Afterbrainisblockedforsectioning,washinseveralchangesofbufferfor2-3hours.
4.SpecimensforEMaresectionedonaVibratome(50μm)andrinsedinbuffer,thoseforLMshouldbecryoprotectedin30%sucroseinbuffer.
5.Afterfreezingwithdryice,30-40μmthicksectionsofLMspecimensarecutonacryostat.
6.Sectionsarerinsed,andthenstoredinphosphatebuffercontaining0.1%sodiumazide.
II)StainingProcedure
Tissueisprocessedasfreely-floatingsectionsincontinuouslyagitatedsolutions.Allincubationsareperformedatroomtemperatureunlessotherwisestated.
1.a.ForlocalizingChAT-positivesomataanddendrites:
Sectionsarewashedin0.1MTris-bufferedsaline(TBS;containing1.4%NaCl,pH7.3)only.Nodetergentorenzymepretreatmentisused.
b.ForlocalizingChAT-positiveterminal-likestructures:
IncubatesectionsinTBS(pH8.1)for5minutesat37°C.TransfersectionstoTBS(pH8.1)containingpronase(1.2μg/mL)for11/2-2minutesat37°C,followedbyseveralicecoldbufferwashesforatotalof5minutes.Theconcentrationofpronaseandincubationtimeofthedigestionshouldbeevaluatedforeachregionexamined.
c.ForlocalizingChATimmunoreactivityandsubsequentlycounterstainingthesections:
IncubationinTBScontaining0.1%-0.8%TritonX-100for15minutesmayincreasethetissuepenetrationoftheImmunoReagents,butitalsoraisesthebackgroundstaining.
2.Incubatesectionsinnormalgoatserum(3-5%)foronehour.Theworkingsolutionsofallantiserashouldalsocontainsimilarlydilutednormalgoatserum.
3.Incubateinanti-ChATmonoclonalantibodysolution(Suggestedworkingdilution1:250,finalworkingdilutionmustbedeterminedbyenduser)for2hoursatroomtemperatureandthenforanadditional6-18hoursat4°C.
4.Incubatewithsecondantibody(i.e.Goatanti-MouseIgG,Cat.No.:AP124,dilution1:50-100)for1-2hours.
5.IncubatewithdilutedPAPcomplex(i.e.MousePAP,CatNo.:PAP14,conc.25-50μg/mL)foronehour.
6.Afterrinsinginbuffer,thesecondantibodyandPAPstepsarerepeatedfor40minutesto1houreachinordertoamplifystainingintensity,particularlyofsmallChAT-containingstructures.
7.Reactfor15minuteswith0.06%3,3"-diaminobenzidine×4HCl(DAB;dilutedinphosphatebufferedsaline,pH7.3)and0.006%H2O2.
8.SpecimensforroutineLMarepostfixedfor1minutesin0.005%OsO4(osmiumtetraoxide),andthenmounted,dehydratedandcoverslipped.SelectedregionsblockedforEMarepostfixedin2%OsO4for1hour,enblocstainedwithuranylacetate,andflat-embeddedinEpon-Aralditeresin. |