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
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
