[Histonet] failure of nuclear counterstaining after IHC

[David Wright]

Hi Histonet & Maria

Is it possible that Maria really means Nissl staining of neuronal cell bodies (somata) together with their nuclei, rather than just nuclei? Tim is right to point out that the staining target is likely getting washed away during IHC processing, but I suggest it is Nissl substance getting degraded, rather than loss of proteins.

Gallocyanine is a nucleic acid stain, and both cresyl violet and (m)ethyl green [are you trying to bait John Kiernan to get a reply?  ;) ] are made relatively specific for nucleic acids when used in an acidic solution (sodium acetate or 1% acetic acid), at which pH only the phosphate backbones of the nucleic acids remain charged and able to bind a variety of stains.

Nucleic acids include both the DNA in the nucleus as well as more widely distributed RNA. The 'Nissl substance' which reveals the neuronal cell bodies is basically the ribosomal RNA and mRNA, which nerve cell bodies have in abundance - at least while they are alive or well preserved. RNA is notoriously quickly degraded once in an aqueous environment, due to the everpresent and hard to inactivate RNases everywhere - ask any molecular biologist.

I do IHC on free-floating 40um brain sections and never get good Nissl staining after incubations, although the Nissl quality on parallel sections not put through IHC is always fine. One problem with thick free-floating sections is the much longer incubation times they require for the same Ab concentration (rule of thumb: increase time with the square of the increased thickness - or half-thickness for floating sections with two accessible cut surfaces).  This longer time in incubation buffer allows plenty of opportunity for RNases to go to work and destroy the Nissl substance. Note that it is the total incubation/wash time for all steps before the Nissl stain that is critical.

Maria's better results after IHC with paraffin sections may simply be that these thinner sections allow much shorter Ab incubations and washes, or perhaps more of the endogenous RNases gets killed by paraffin embedding? Any way, my guess is that successful Nissl staining on the 60um sections will require much shorter incubations beforehand. With thick section IHC, that will mean jacking the Ab concentration to dizzying and expensive levels. Immunofluorescence has the advantage of much shorter total incubations, so may help. You could play with RNase inhibitors to see if that helps, but what I do is to double immunostain for a neuronal antigen (such as NeuN) as well. You have experience in double staining and these epitopes do not degrade so easily as RNA.

I'd like to hear what you get to work!

Best wishes - David
==
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026

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ORIGINAL MESSAGES
Histonet Digest, Vol 146, Issue 2 Message: 2
Date: Sun, 3 Jan 2016 20:28:58 -0800
From: Maria Mejia <mbmphoto at gmail.com>
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

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
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Histonet Digest, Vol 146, Issue 2 Message: 4
Date: Mon, 4 Jan 2016 15:54:12 +0000
From: "Morken, Timothy" <Timothy.Morken at ucsf.edu>
To: Maria Mejia <mbmphoto at gmail.com>
Cc: Histonet <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC!

Maria,

If the counterstain is good when done before IHC stain and poor after it sounds like proteins are being extracted during the IHC processing and staining. Have you tried staining sections after each step of the IHC process to isolate the point the stain becomes weak?

Tim Morken
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center