[Histonet] Good TUNEL staining but very few Active Caspase-3 positive cells!!!

[Guillermo Palao]

Hello histoneters,
   
  I am studying apoptosis in mouse kidney samples, and while TUNEL staining looks pretty good (positive cells checked by morphology with DAPI), staining with Anti-Active-Caspase-3 Ab from Cell Signaling shows very low number of positive cells. 
   
  I have tried frozen sections fixed with Acetone or Paraformaldehyde as well as parafin embeded sections boiled in citrate buffer for 10 min. Positive cells look very good, with nice cytoplasmic staining, but I would have expected a higher number of positive cells (See J Pathol 2003; 199: 221–228 for a comparison of TUNEL and Active Caspase-3 staining). I am quite sure that TUNEL staining is specific as I titrated TdT concentration and nuclei look apoptotic with DAPI. How could I improve Active Caspase-3 staining?
   
  Any ideas are most welcome!
   
  Guillermo

		
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