InSituCellDeath(Apoptosis)DetectionbyTUNELlabelingbyBoehringerMannheim(CatalogNo.1684809),modifiedbyJosiahN.WilcoxandJoséC.Rodriguez-EmoryUniversity-April1996ProtocolforFrozenSections:
- Warm150ml4%Paraformaldehyde/1xPBStoRT.Fixslidesinit,20min.,RT.
- 1xPBSrinse,2times.
- 1xPBS,30min.,RT.BeginchillingTriton/SSConice.
- 0.1%Triton/0.1%SodiumCitrate,2min.,4°C.
- Allslides:1xPBSrinse,2times(+10minforthosenon-pos.controlslides).
- (Pos.controlslide:inDNaseIsolution(100µlof200µg/ml),10min.,RT.1xPBSrinse,2timesinaseparatecontainerthencombinewithotherslides.)
- Wipearoundtissue.
- MakeupnegativeControlsolution(justLabelsolutioncontainingFITC)andTUNELsolutionsattimeofuse:
A.Remove100µlfromTube2(Labelsolution)for2negativecontrols(50µleach).Dothisevenifyouareomittingthisnegativecontrolsothatvolumesandconcentrationswillremainconsistentforthelabeling.
B.AddTotalvolume(50µl,)ofTube1(TdT)+remainderofTube2(450µl).
- Apply100µlTUNELreactionmixture(or100µlControlLabelsolutionfornegativecontrol)toeachslide.
- Incubateinhumidchamber,60min.,37°C.
- 1xPBSwash,3times.
- Wipearoundtissue.
- Apply100µlanti-FITC-APconj.("converter-AP")oneachsample.
- Incubateinhumidchamber,30",37°C.
- 1xPBSwash,3times.
- 100mMTrisbuffer,pH8.2,5min.,RT.
- Add50-100µlsubstratesolution(5-6dropsVectorBlueorVectorRedsubstrate/perslide):
- Mix:
5ml100mMTris,pH8.21dropLevamisole2dropseachofSolution1,2,and3ofeitherVectorsubstrate
- Incubateinabsenceoflight,RT.VectorBlue-10min.;VectorRed-20min.
- dH2O,1timetostopcolorreaction.
- CounterstainforVectorBlue:
- Gill"sHematoxylin,No.2,5sec.WaterrinseuntilclearScott"ssolution,20sec.Waterrinseuntilclear70%EtOH,30sec.95%EtOH,2times,30sec.each100%EtOH,2times,30sec.eachHistoclear,1min.Histoclear,1min.CoverslipwithAccumountmedium.
- CounterstainforVectorRed:
- Gill"sHematoxylin,No.2,5sec.WaterrinseuntilclearScott"ssolution,20sec.Waterrinseuntilclear70%EtOH,30sec.95%EtOH,2times,30sec.each100%EtOH,2times,30sec.eachXylenes,1min.Xylenes,1min.CoverslipwithAccumountmedium.
- ReagentsNeeded:
- 3L1xPBS
10mMTris-HCl
20mg/mlProteinaseK
100µl/slidex-phosphate/BCIPorFastRedsubstrate
DNaseIsolution(1mg/ml-1µg/ml)forPositivecontrol
100mMTris-Hcl,pH8.2
4%Paraformaldehyde/PBS,pH7.4
- Heat500ml1xPBSto65°C.
- Dissolve20gParaformaldehydeinthePBS(inafumehood).
- FilterwithWhatmanNo.1papertoremoveparticles;storeat4°C.
0.1%TritonX-100in0.1%SodiumCitrate(SSC)
- Mix:
2ml20xSSC400µlTritonX-100398mlH2O
100mMTris-Hcl,pH8.2
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