We have some old mouse brains stored in 4% PFA for several years. DAPI and Hoechst stains are not working on this tissue, athough immunohistochemistry for GFAP is OK (after antigen retrieval). What's going on that the DAPI doesn't work? Long term PFA fixation causing DNA degradation?
Hi Catherine A Gorrie
Yes, long term PFA fixation is not advised if you want to study DNA or Nucleus as it can cause DNA loss and denaturation.
- DNA extraction/denaturation: Long PFA fixation can cross-link and denature DNA, making it less accessible to DAPI binding.
- Loss of cellular integrity: Extended fixation can weaken cell membranes, leading to leakage of DNA and reduced staining intensity.
Post sacrifice and brain isolation, It is recommended to keep them for 1-2 days at 4 degree and then it can be stored in 30% sucrose for long term storage (For cryotome sections).
Thanks,
We had problem once on PFA-fixed slices. It was recommended that before adding DAPI, let the section almost dry (not full dry). Also dilute fresh DAPI from frozen concentrate aliquotes (1000x) . Do not re-freeze aliquotes of DAPI and re-use.
As Samir mentioned above, longer time of PFA fixation can be a serious problem. Good luck
Intermediate filaments, like GFAP filament, are tougher than DNA. With time, the paraformaldehyde breakdown produces formic acid and the DNA and many proteins are damaged. DAPI binds to the minor groove of AT rich sequences, so if that conformation is lost, staining is lost. Storage at -20 in ethanol after fixation is better long term.
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