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

[WILLIAM DESALVO via Histonet]

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.

Sent from my Windows Phone

From: Morken, Timothy via Histonet
Sent: ‎1/‎4/‎2016 9:20 AM
To: Maria Mejia
Cc: Histonet
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@lists.utsouthwestern.edu]
Sent: Sunday, January 03, 2016 8:29 PM
To: histonet@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] Help with causes or theory behind failure of nuclear counterstaining after IHC! (Maria Mejia)

[Steven Weston via Histonet]

Maria,
This may sound simplistic but I find that if I have problems such as this the 
first thing I do is try a new batch of the staining reagents. If you haven't 
had problems before then something must have changed either in your protocols 
or your reagents. It could be that your heat retrieval reagents are too old or 
contain detergents that remove some of the proteins you are looking for. Some 
of the proprietary heat retrieval reagents that allow you to heat retrieve 
without having to dewax  by going through xylene have been shown to change the 
nuclear staining pattern and create what appear to be nuclei that are full of 
vacuoles.
Also if the celloidion is not completely removed during your staining it may be 
stopping any of the higher molecular weight stains from penetrating the cells. 
Try leaving your sections in acetone for a number of changes to ensure full 
removal of the celloidion.
Regards
Steve Weston
University of Tasmania
Breathe-Well CRE
Lab Manager
0408990859




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

[Morken, Timothy via Histonet]

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@lists.utsouthwestern.edu] 
Sent: Sunday, January 03, 2016 8:29 PM
To: histonet@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] Help with causes or theory behind failure of nuclear counterstaining after IHC!

[Maria Mejia via Histonet]

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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