I'm trying to run an assay for Caspase 3/7 activity to visualize apoptotic cells in flash frozen, unfixed mouse skin sections. The assay involves a non-fluorescent reagent that is processed into green fluorescence by endogenous, active Caspase 3 or 7 in apoptotic cells.
After thawing the slides for 3 minutes and washing once in PBS to remove OCT, I added the reagent to the unfixed tissue (I've tried 1 hour at room temp, or 30 mins at 37C). After the incubation, I post-fixed the sections with 4% paraformaldehyde, added DAPI mounting medium then visualized. 100% of the cells had green fluorescence, and the nuclei appeared enlarged so it looks like the cells are bursting or autolysing during the incubation. I'm wondering if I should fix the tissue immediately after thawing to stop the damage to the cells.
What would be the best method for post-fixing flash frozen mouse skin so that endogenous enzymatic activity is maintained? Does anyone have experience specifically with using the CellEvent Caspase 3/7 assay from Invitrogen in frozen tissue sections?
PS. I have also tried a TUNEL assay which generated no signal.
I would suggest you to carry out ELISA or WB analysis of Caspases 3/7 for the detection of apoptosis in flash frogen tissue. If you fix the tissue, the enzyme activity will be lost, hence you can homogenize the tissue and take the sup to anayse capase-3/7 activity using ELISA and level by WB.
As you mentioned in your content, you can go ahead with the TUNEL Assay that is very sensitive ,(For example R & D Systems USA) and you can analyse DNA fragmentation in a single cell of you sectioned tissue.
Best
Hi Fiona,
Maybe you could try something like the protocol in the paper here.
Good luck with your experiment!
Aja
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