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

[WILLIAM DESALVO]

Maria, I. Think you may have a pH issue. High pH results in reduction of protons, H+, effects dye structure and can cause light to no staining after bluing. If you are using hematoxylin w/ aluminum, most popular, decreased pH = decreased intensity. Acid breaks the Al+3. Check your ph throughout the process. Sounds like something has changed. Good luck.

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________________________________
From: Morken, Timothy via Histonet<mailto:histonet at lists.utsouthwestern.edu>
Sent: ‎1/‎4/‎2016 9:20 AM
To: Maria Mejia<mailto:mbmphoto at gmail.com>
Cc: Histonet<mailto: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



-----Original Message-----
From: Maria Mejia via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Sent: Sunday, January 03, 2016 8:29 PM
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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