Hello,
I am performing an immunofluorescence TUNEL assay using an abcam kit (ab66110) on frozen tissues of kidney mice using the following protocol from abcam (frozen tissues section) https://www.abcam.com/ps/products/66/ab66110/documents/tunel-assay-kit-protocol-book-v12-ab66110%20(website).pdf. I want to assess apoptosis in podocytes at 24hrs post-irradiation. For that purpose I am also staining for podocin with secondary Alexa Fluor 488 following the TUNEL assay.
My issue is that I am getting false-positives for TUNEL staining in both control and irradiated conditions. Most of the cells seem to be light up. The podocin antibody is specific and only observable in glomeruli which means there is no problem regarding fixation or permeabilization with the standard staining protocol that I use. However I don’t understand why I am having such a strong TUNEL background.
Is this related to the proteinase K step? Should I increase the time for this one ? I usually do this step for 5min. I have seen that proteinase K digests endogenous nucleases, maybe my section are not getting enough of time for that ?
BrdU-Red has an excitation peak at 488 that is an overlap with 488 nm Alexa Flour 488, not the great choice of chromophores to combine in the same image. Another thing the BrdU-Red staining is very unstable and should be imaged within 1-3 hours after the staining is completed. This kit is primarily for flow cytometry analysis rather than IHC and it is definitely not meant to be combined with antibody staining. I would recommend switching to cleaved caspase-3 if you want to see the apoptosis combined with podocin staining.
Hi Patrick. Take a look at this work, I hope it helps!
Article False positive staining in the TUNEL assay to detect apoptos...
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