Hi there,
We are working with GeoMx (Nanostring) and we are facing some problems regarding SYTO13 nuclear staining with FFPE blocks prepared with Sakura X-Press 120 and Sakura Autotec 120 . It seems that this automated preparation of the blocks is interfering somehow with nuclear staining and following segmentation and collection of regions of interest. Staining pattern doesn't seem specific, more like background (see attached image). It is as SYTO13 is not able to enter the cell??
Anyone facing the same problem or any ideas, please?
Thanks in advance :)
Hey Helena Suarez !
It’s probably because of the glyoxal fixation messing with DNA accessibility and maybe even some leftover clearing agents that block the stain. A few troubleshooting tips could be like making sure that dewaxing is extra thorough (like longer xylene steps), tweaking antigen retrieval a bit (higher pH or shorter enzyme time), and even bumping up the SYTO13 concentration or giving it more time to stain. You could also try an RNase treatment to see if most of the background is just RNA. If you’ve got a formalin-fixed sample to compare, that’ll confirm if it’s a processing issue. Hope something clicks — let me know how it goes! Cheers!!
Many thanks for your recommendations, we'll try first to use an antigen retrieval solution pH 6 instead of 9 and see what happens :)
We know for sure that it is something related to the processing issue before our steps in the lab, we have done it several times with FFPE blocks prepared with another method (not Sakura) and they work perfectly on our hands with the same protocol and reagents.
Best regards
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies