[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[Maria Mejia]

Happy New Year Everyone,

I'm the lead histologist working in an IHC based research lab focused on early stages
of Alzheimer's disease.  We work on paraffin sections processed & cut from 600um celloidin 
sections.  Including a lot of 60um cellodin sections from whole human brainstem.  

For years, everything has going good regarding counterstaining after single & double IHC staining
on 60um free-floating sections.  However for the past two months we've struggled to achieve
good visible counterstaining on IHC sections to count the stained neurons - to see clearly
the nucleus & nucleolus!  

For a number of years, Gallocyanine was our choice of counterstain after IHC.  Now, it's
NOT working (neurons not stained visible enough to count).  We've also tried cresyl violet
counterstain -  staining too weak!   In both counterstains, we modified
the staining protocols quite a number of times to get good visible staining - nothing!!!

Strangle because we get lovely counterstained neurons with NO IHC staining on our 60um
sections, but as soon as we take the sections through the IHC protocol e.g. antigen retrieval, 
antibodies & chromogens - counterstain is too weak!  Our paraffin IHC sections work & look 
wonderful!

Now, my PI wants to try methyl green counterstain, however I think we'll have the same problem.
Here's what I need help with:

1) Can someone please explain the reason or theory  behind the failure of counterstain uptake
by cells such as human neurons on 60um celloidin sections?

2) Can anyone please offer staining protocols that use alternative dehydration & clearing reagents.
I've been using alcohols dehydration (96% & 100%) without success as well as clearing with xylene - 
which hardens the tissue if left too long in this reagent. 

 I was thinking of perhaps using acetone instead of alcohols & maybe using a methyl salicylate or
chloroform.  Thoughts anyone?

I wish Dr Chris van der Loos was still with us.  I'd dearly like to hear from anyone who can help with
this issue - Gayle Callis, Terry Johnson, Dr Hohn Kiernan et al.

Any assistance anyone can provide will be greatly appreciated!

Best
Maria Mejia
UCSF
Memory & Aging Department
San Francisco, CA