Hello everyone, I am asking this question as I have found no solutions for this on any other ResearchGate threads or published literature.
I am using the Roche In-situ cell death detection kit (Fluoroscein), and combining it with conventional antibody based immunofluorescence protocol. I work with free-floating sections and conduct antibody staining first, followed by slide mounting onto gelatin subbed slides, briefly drying the sections to avoid detachment during labeling and washing, conducting the TUNEL reaction at 37º C for 1h (have tried 1.5 hrs as well), washing 3 x 15 minutes in buffers and coverslipping. I will attach my full protocol as well as sample images of the results. I also tried a modified protocol (https://www.researchgate.net/post/Problems_with_fluorimetric_TUNEL_assay_in_free_floating_brain_sections2) as described in this link and in my protocol including step of incubating in PBST at 85º C prior to TUNEL labeling, which improved the brightness of the staining.
The issue is that in stroke brains, apoptosis should occur in the peri-infarct region containing cells earlier in apoptosis/cell death, while the infarct region should be largely unstained due to the cells undergoing severe necrosis. I see most of the TUNEL staining in infarct area, which would be ok if there was any significant amount of labeling in the peri-infarct zone. Another minor problem is that I have some small tissue "microfolds" that take up stain more brightly than any apoptotic cell, but that I think should be fixed with better technique and less tissue drying prior to labeling.
Is there a protocol for doing TUNEL stain in free-floating sections before mounting them onto slides? Is there any better kit out there that is more sensitive and brighter labelling?
List of other things I have tried:
Cryostat sectioning at 20-30 µM. (Current samples discussed are 40 µM sections from a freezing microtome)
Antigen retrieval in citrate buffer O/N before antibody staining. (60º C)
Antigen retrieval in citrate after Ab staining before TUNEL (60º C, and microwave)
Doubling the Tdt concentration
Proteinase K before TUNEL
citrate, followed by Tris-HCl before TUNEL (recommended in Roche manual)
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