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电热恒温水浴锅电热恒温水浴锅批发、促销价格、产地货源
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  1. Sectionsofparaformaldehydefixed,OCT-embeddedvasculartissuearesectionedat7to10mmandstored,desiccatedat-70°Cuntilneeded.
  2. Thawslidesimmediatelybeforeuse,usually10-30min.priortobeginningtheprotocol.
  3. Fixinacetone5min.,airdryforapproximately20min.,RT.
  4. ImmerseinProteinaseK/1xPBSfor10min.,RT(7.5µlProteinaseKof20mg/mlstockin150ml1xPBSforafinalconcentrationof1µg/ml).
  5. Rehydratein1xPBStwicefor5min.each,RT.
  6. Immersein4NHClfor10min.,RT.
  7. Immersein1xTBE,pH8.4for5min.,RT.CheckpHofbufferwithpHpapertoseeifitisneutral.
  8. Immersein1xPBS.CheckpHofbufferafter2min.ApplyprimaryantibodywhenpHis7.0-7.5.
  9. Preparetheworkingdilutionoftheprimaryantibodyin1.0%crystalline-gradeBovineSerumAlbum(BSA)in1xPBS.UseBrdUantibody(DakoCatalogNo.M744)at1:20dilution.BlotoffPBSandapply150µlofworkingdilution.Incubatesectionsinahumidchamberfor1hour,RT.Blotoffantibody,wash2times5min.eachin1xPBS.
  10. Preparetheworkingdilutionofthesecondaryantibody(Horseanti-mouseIgG-VectorCatalogNo.BA2001)in1.0%BSA/PBS,2.0%normalhorseserum.Preparethesecondaryantibodyata1/400dilution.Apply150µlandincubate30min.,RTinahumidchamber.Blotofftheantibody,wash2times5min.eachin1xPBS.
  11. PreparetheworkingdilutionofABC-VectorRedcomplexfromtheVectorABCkit(CatalogNo.AK-5000)beforefinishingthepreviousstep.Mix5mlPBS,twodropsofSolutionA,andtwodropsofSolutionB,andallowtositat4°Cfor30min.priortouse.Applyenoughtocoverthesectionsontheslide,andincubatefor1hour.Blotoffthesolution,wash2times5min.eachin1xPBS,followedby1washin100mMTrispH8.2for5min.
  12. MakeupVectorRedsubstratesolution(CatalogNo.SK-5100)immediatelybeforeuse.Incubateinthedarkfor20-30min,stopcolorreactionbyblottingoffsubstrateandrinsingindH2Ofor5-10min.
  13. Counterstainwithhematoxylin,1%acid-alcohol,Scott"ssolution,dehydratethroughgradedalcohols,thenxylenesperprotocolbelow,andcoverslipforviewing.
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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