RE: [Histonet] Re: Good TUNEL staining but very few Active Caspase-3 positive cells

From:Guillermo Palao

  What you say seems to make perfect sense in terms of kinetics of apoptosis, but actually in the paper I found comparing both methods the result was the opposite (ie. Greater number of Active Casp-3+  than TUNEL+ cells). However the authors did not postulate any possible mechanism to explain this... 
  Could you send more details on the pepsin digestion you used to improve sensitivity of Ab? Did you try Proteinase K treatment of samples?
  Thanks for your input,

"Johnson, Teri"  escribió:

Good question! I'll take a stab at it and will read with interest any
additional commentary on this.

We found to get good staining with Cleaved Caspase-3 in paraffin section
we needed to add a brief pepsin digestion in addition to the HIER to
achieve staining. Without it we found we needed to use tyramide
enhancement to see it in fluorescence. I think the antibody really does
exhibit sensitivity issues.

Otherwise, caspase-3 is activated early in the apoptotic process. It
could be that it is no longer in its activated state in later stages of
apoptosis, whereas the TUNEL will show positive across a wide range of
apoptosis stages.

I'm an amateur on the mechanism of this event, and welcome Dr. Cartun or
anyone with more advanced knowledge of this to confirm or disagree with
my conjecture.

Best wishes,

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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