[Histonet] Samples not staining with Hematoxylin or primary

[Anna Huntley Coffey]

Thanks so much to everyone who responded! A couple people have suggested
that this issue may be due to a disruption to the charge on the slide (due
to high temp baking, water bath additives, etc) which causes a hydrophobic
barrier to form and prevents reagent from reaching the tissue. This sounds
very convincing to me, but we have found that repeated antigen retrieval
using a pressure cooker (our standard retrieval for this particular
primary) has allowed 2 of the 3 slides to stain with both DAB and
Hematoxylin. Would repeated retrieval be able to reverse the slide charge
back to normal?


I'm having an issue with some recent samples and was wondering if anyone
else has experienced the same thing...I stained some samples with a
previously optimized protocol for anti-human Granzyme B on the Ventana
Benchmark and about 1/3 of my samples did not stain at all with either
GranzB or Hematoxylin. My controls and samples that did stain positive for
GranzB look fine and the machine has been recently serviced and working
well with other runs. I'd really appreciate your input if you have any
advice or suggestions on what could have gone wrong.

Thanks,
Anna