供应 陕西超纯水机,延安超纯水机,咸阳超纯水机「纯水制取设备」马 ...
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Sectionsofparaformaldehydefixed,OCT-embeddedvasculartissuearesectionedat7to10mmandstored,desiccatedat-70°Cuntilneeded.Thawslidesimmediatelybeforeuse,usually10-30min.priortobeginningtheprotocol.Fixinacetone5min.,airdryforapproximately20min.,RT.Immersein0.3%H2O2/Methanolfor15min.,RT.(450µlH2O2in150mlMethanol).ImmerseinProteinaseK/1xPBSfor10min.,RT(7.5µlProteinaseKof20mg/mlstockin150ml1xPBSforafinalconcentrationof1µg/ml).Rehydratein1xPBStwicefor5min.each,RT.Immersein4NHClfor10min.,RT.Immersein1xTBE,pH8.4for5min.,RT.CheckpHofbufferwithpHpapertoseeifitisneutral.Immersein1xPBS.CheckpHofbufferafter2min.ApplyprimaryantibodywhenpHis7.0-7.5.Preparetheworkingdilutionoftheprimaryantibodyin1.0%crystalline-gradeBovineSerumAlbum(BSA)in1xPBS.UseBrdUantibody(DakoCatalogNo.M744)at1:20dilution.BlotoffPBSandapply150µlofworkingdilution.Incubatesectionsinahumidchamberfor1hour,RT.Blotoffantibody,wash2times5min.eachin1xPBS.Preparetheworkingdilutionofthesecondaryantibody(Horseanti-mouseIgG-VectorCatalogNo.BA2001)in1.0%BSA/PBS,2.0%normalhorseserum.Preparethesecondaryantibodyata1/400dilution.Apply150µlandincubate30min.,RTinahumidchamber.Blotofftheantibody,wash2times5min.eachin1xPBS.PreparetheworkingdilutionofABC-PeroxidasecomplexortheVectorABC-peroxidaseElite(CatalogNo.PK-6100)beforefinishingthepreviousstep.Mix5ml1xPBS,twodropsofSolutionA,andtwodropsofSolutionB,andallowtositat4°Cfor30min.priortouse.Applyenoughtocoverthesectionsontheslide,andincubatefor1hour.Blotoffthesolution,wash2times5min.eachin1xPBS,followedby1washin100mMTrispH8.2for5min.Makeupsubstratesolutionimmediatelybeforeuse.Add5mlofsterilewater,twodropsofbufferfromVectorDABsubstratekit(CatalogNo.SK-4100),fourdropsDAB,twodropsH2O2,andtwodropsofNickelfromkit,in15mltube(wrapthetubeinfoiltoprotectthesubstratefromlight).Incubateinthedarkfor20-30min.,stopcolorreactionbyblottingoffsubstrateandrinsingindH2Owaterfor5-10min.Caution:DABishighlycarcinogenic,sohandlewithglovesandworkonabsorbenttowels.Inactivatesolutionsbypouringintoabeakercontaining3%KMnO4and2%Na2CO3inwater,andthendisposedownsink.Counterstainwithhematoxylin,1%acid-alcohol,Scott"ssolution,dehydratethroughgradedalcohols,thenxylenesperprotocolbelow,andcoverslipforviewing.- Hydrogenperoxide
- SIGMACatalogNo.H-1009(5ml)
- ProteinaseK
- DissolveProteinaseK(SigmaCatalogNo.P-4914)indH2Oforaconcentrationof20mg/ml.
- 1%BSA/PBS
- 1gFractionVBSAin100ml1xPBS.Mixinabeakerwithlowheat.Aliquotin15mltubesandstoreat-20°C.
- Hematoxylin
- 75mlGill"shematoxylinno.2in75mldH2O.
- 1%Acid-alcohol
- 2mlHClin198mlof70%ethanol.
- Scott"sSolution
- NaHCO3,2g
- MgSO4.7H2O,20g
- dH2O,1000ml
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