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

[Johnson, Teri]

Guillermo,

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