[Histonet] Collodian bag for cell blocks

[Baldwin, Kathy via Histonet]

Hi All
Was wondering if anyone is using the Collodian bag for cell blocks.  We just 
got it and have been using it 'works great' but all the techs are complaining 
of the smell.. Our ventilation has been looked at and has passed however the 
smell still lingers in the room.  Any suggestions??


Thanks
S. Kathy Baldwin
Histology/Cytology Supervisor
Memorial Hospital and Health Care Center
800 West 9th St.
Jasper, Indiana  47546
Office 812-996-0210
Fax 812-996-0232
Cell 812-887-3357





CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for 
the sole use of the intended recipient(s) and may contain confidential and 
privileged information or otherwise protected by law. Any unauthorized review, 
use, disclosure or distribution is prohibited. If you are not the intended 
recipient, please contact the sender by reply e-mail and destroy all copies of 
the original message.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[Histonet] IHC/BAL slides

[Lager, Loree via Histonet]

Hello All,

Our pathologist is requesting a couple of IHC stains on air dried BAL slides, 
for interest.  We currently only run IHC's on paraffin and frozen tissue.

If anyone has  protocols for CD3  and CD56, including any fixation, using the 
Benchmark Ultra and Ventana iView DAB detection kit, I would greatly appreciate 
it.

Thanks,
Loree L Williams
Seattle Children's Hospital
Histology
CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for 
the sole use of the intended recipient(s) and may contain confidential and 
privileged information protected by law. Any unauthorized review, use, 
disclosure or distribution is prohibited. If you are not the intended 
recipient, please contact the sender by reply e-mail and destroy all copies of 
the original message.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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

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

Re: [Histonet] Collodian bag for cell blocks

[Rene J Buesa via Histonet]

Do you mean "collodion"? If so it is nitrocellulose, HIGHLY flammable and even 
explosive. Has a characteristic "pungent" odor.Highly unsafe and it is beyond 
me why would you use this product. The odor can "adhere" to clothing.You may 
have a ventilation system that "passes inspection" but if your techs can smell 
it, it is there and it should not be.Have you consulted the MSDS for this 
chemical?I would not use it, no matter how great it works.René 

On Monday, January 4, 2016 4:07 PM, "Baldwin, Kathy via Histonet" 
 wrote:
 

 Hi All
Was wondering if anyone is using the Collodian bag for cell blocks.  We just 
got it and have been using it 'works great' but all the techs are complaining 
of the smell.. Our ventilation has been looked at and has passed however the 
smell still lingers in the room.  Any suggestions??


Thanks
S. Kathy Baldwin
Histology/Cytology Supervisor
Memorial Hospital and Health Care Center
800 West 9th St.
Jasper, Indiana  47546
Office 812-996-0210
Fax 812-996-0232
Cell 812-887-3357





CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for 
the sole use of the intended recipient(s) and may contain confidential and 
privileged information or otherwise protected by law. Any unauthorized review, 
use, disclosure or distribution is prohibited. If you are not the intended 
recipient, please contact the sender by reply e-mail and destroy all copies of 
the original message.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet


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

Re: [Histonet] Collodian bag for cell blocks

[WILLIAM DESALVO via Histonet]

What method are you using? Making your own coated tubes, UCSF or MD Anderson. 
Are you purchasing coated tubes? Once the tube is coated, dried (using hood) 
and filled w dH2O, there should be no odor. Always work w/ collodion under the 
hood. Ethyl ether is main component. Once in cassette and processed, there 
should not be odor.

Sent from my Windows Phone

From: Baldwin, Kathy via Histonet
Sent: ‎1/‎4/‎2016 2:07 PM
To: 
'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Collodian bag for cell blocks

Hi All
Was wondering if anyone is using the Collodian bag for cell blocks.  We just 
got it and have been using it 'works great' but all the techs are complaining 
of the smell.. Our ventilation has been looked at and has passed however the 
smell still lingers in the room.  Any suggestions??


Thanks
S. Kathy Baldwin
Histology/Cytology Supervisor
Memorial Hospital and Health Care Center
800 West 9th St.
Jasper, Indiana  47546
Office 812-996-0210
Fax 812-996-0232
Cell 812-887-3357





CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for 
the sole use of the intended recipient(s) and may contain confidential and 
privileged information or otherwise protected by law. Any unauthorized review, 
use, disclosure or distribution is prohibited. If you are not the intended 
recipient, please contact the sender by reply e-mail and destroy all copies of 
the original message.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

[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




University of Tasmania Electronic Communications Policy (December, 2014).
This email is confidential, and is for the intended recipient only. Access, 
disclosure, copying, distribution, or reliance on any of it by anyone outside 
the intended recipient organisation is prohibited and may be a criminal 
offence. Please delete if obtained in error and email confirmation to the 
sender. The views expressed in this email are not necessarily the views of the 
University of Tasmania, unless clearly intended otherwise.
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Re: [Histonet] failure of nuclear counterstaining after IHC

[David Wright via Histonet]

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


ORIGINAL MESSAGES
Histonet Digest, Vol 146, Issue 2 Message: 2
Date: Sun, 3 Jan 2016 20:28:58 -0800
From: Maria Mejia 
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.  

[Histonet] Height regulations

[Anne Murvosh via Histonet]

Putting in some lower shelving for storage and I can't remember the height 
regulation for keeping stuff off the floor.  I tried to google it, it must be 
buried in some government doc.  Please Help?  Thanks Anne
___
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet

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

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