IMMUNOCYTOCHEMICALIDENTIFICATIONOFPROLIFERATINGCELLSINVASCULARTISSUE(ANTI-BrdU);VectorRedDetectionJosiahN.WilcoxandRomeshR.Subramanian-EmoryUniversity-September20,1993- Sectionsofparaformaldehydefixed,OCT-embeddedvasculartissuearesectionedat7to10mmandstored,desiccatedat-70°Cuntilneeded.
- Thawslidesimmediatelybeforeuse,usually10-30min.priortobeginningtheprotocol.
- Fixinacetone5min.,airdryforapproximately20min.,RT.
- 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-VectorRedcomplexfromtheVectorABCkit(CatalogNo.AK-5000)beforefinishingthepreviousstep.Mix5mlPBS,twodropsofSolutionA,andtwodropsofSolutionB,andallowtositat4°Cfor30min.priortouse.Applyenoughtocoverthesectionsontheslide,andincubatefor1hour.Blotoffthesolution,wash2times5min.eachin1xPBS,followedby1washin100mMTrispH8.2for5min.
- MakeupVectorRedsubstratesolution(CatalogNo.SK-5100)immediatelybeforeuse.Incubateinthedarkfor20-30min,stopcolorreactionbyblottingoffsubstrateandrinsingindH2Ofor5-10min.
- 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
- Hematoxylin-10sec.
- rinseinwateruntilwaterisclear
- Veryquickdipin1%acid-alcohol(<1sec.)
- rinseinwater
- Scott"ssolution-20sec.
- rinseinwater
- Dehydratethroughgradedalcohols(70%,95%,95%,100%,100%)
- Xylenes
- Coverslip
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