[Histonet] help

Sun Apr 26 14:16:58 CDT 2009

Hello, I have a friend who is having trouble with ziehl-neesen stainin. Her sections appear red (with no blue). I have a feeling she may not be destaining adequately. Any suggestions?

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Sent: Sun 4/26/2009 12:05 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 65, Issue 44

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Today's Topics:

   1. paraffin embedding (Marc Shaeffer)
   2. Long term storage for IHC? (Mikael Niku)
   3. Re: Long term storage for IHC? ( TF )
   4. AW: [Histonet] Long term storage for IHC? (Gudrun Lang)
   5. Long term storage for IHC ? (Rene J Buesa)

----------------------------------------------------------------------

Message: 1
Date: Sat, 25 Apr 2009 10:15:50 -0700
From: "Marc Shaeffer" <mshaeffer <@t> cox.net>
Subject: [Histonet] paraffin embedding
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <B7392E4768D3431D997FB5694D0C7FC5 <@t> youro0kwkw9jwc>
Content-Type: text/plain;	charset="us-ascii"

Don't forget to put the plastic cassette on top the mold prior to filling
and solidifying the paraffin.

------------------------------

Message: 2
Date: Sat, 25 Apr 2009 23:24:56 +0300
From: Mikael Niku <mikael.niku <@t> helsinki.fi>
Subject: [Histonet] Long term storage for IHC?
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <49F37198.5030907 <@t> helsinki.fi>
Content-Type: text/plain; charset=ISO-8859-1; format=flowed

Dear Histonetters,

what do you think is the best way to store formalin-fixed tissues for 
immunohistochemistry, long-term (several years)?

We are currently fixing overnight, then rinsing in buffer and storing in 
70% or 100% ethanol, +4C or -20C. However, I don't really KNOW what 
happens here and how the tissues will work after the years. I guess 
basically the formalin fixation will be at least somewhat reversed and 
after some time the tissues will be more like ethanol fixed. But are 
there better options? Long term storage in formalin will make IHC 
difficult. And this is about large numbers of tissue samples, only some 
of which will be actually used later, so we wouldn't like to process all 
of them to paraffin either.

With best regards,
Mikael Niku
University of Helsinki, Finland

------------------------------

Message: 3
Date: Sun, 26 Apr 2009 13:19:53 +0800
From: " TF " <tifei <@t> foxmail.com>
Subject: Re: [Histonet] Long term storage for IHC?
To: " Mikael Niku " <mikael.niku <@t> helsinki.fi>, " histonet "
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200904261319483656380 <@t> foxmail.com>
Content-Type: text/plain;	charset="utf-8"

Hi, i embeded all of them in OCT and put them under -20.
To prevent water vapor leakage, use some parafilm to pack the OCT block or seal it.
We tested this in samples harvested 5 years ago (2004-2009).

2009-04-26 

TF 

å'ä»¶äººï¼s Mikael Niku 
å'é?æ-¶é-´ï¼s 2009-04-26  04:31:05 
æ"¶ä»¶äººï¼s histonet 
æS"é?ï¼s 
ä¸»é¢~ï¼s [Histonet] Long term storage for IHC? 

Dear Histonetters,
what do you think is the best way to store formalin-fixed tissues for 
immunohistochemistry, long-term (several years)?
We are currently fixing overnight, then rinsing in buffer and storing in 
70% or 100% ethanol, +4C or -20C. However, I don't really KNOW what 
happens here and how the tissues will work after the years. I guess 
basically the formalin fixation will be at least somewhat reversed and 
after some time the tissues will be more like ethanol fixed. But are 
there better options? Long term storage in formalin will make IHC 
difficult. And this is about large numbers of tissue samples, only some 
of which will be actually used later, so we wouldn't like to process all 
of them to paraffin either.
With best regards,
Mikael Niku
University of Helsinki, Finland
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Histonet <@t> lists.utsouthwestern.edu
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------------------------------

Message: 4
Date: Sun, 26 Apr 2009 12:46:50 +0200
From: "Gudrun Lang" <gu.lang <@t> gmx.at>
Subject: AW: [Histonet] Long term storage for IHC?
To: "'Mikael Niku'" <mikael.niku <@t> helsinki.fi>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <7224DDE4D9664677A8281EECFDDD9309 <@t> dielangs.at>
Content-Type: text/plain;	charset="iso-8859-1"

I guess the formalin-fixation will be reversed although in a very slow
manner in the 70% ethanol. And I think, overnight fixation depending on the
tissue size will give insufficient formalin-fixation. This will result in
ethanol-fixation in the 70% and especially in the 100% ethanol.
If you want long time storage in ethanol, be sure that formalin-fixation is
sufficient. 
In my opinion your regular immunhistoprotocol has also to be adapted after
long time storage (more than 2 months), no matter if in formaldehyd or
ethanol.
What is more effort? Storage and administration of large numbers of wet
tissue in refrigerator or processing to paraffin blocks and storage in
drawers?

Gudrun Lang

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Mikael
Niku
Gesendet: Samstag, 25. April 2009 22:25
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] Long term storage for IHC?

Dear Histonetters,

what do you think is the best way to store formalin-fixed tissues for 
immunohistochemistry, long-term (several years)?

We are currently fixing overnight, then rinsing in buffer and storing in 
70% or 100% ethanol, +4C or -20C. However, I don't really KNOW what 
happens here and how the tissues will work after the years. I guess 
basically the formalin fixation will be at least somewhat reversed and 
after some time the tissues will be more like ethanol fixed. But are 
there better options? Long term storage in formalin will make IHC 
difficult. And this is about large numbers of tissue samples, only some 
of which will be actually used later, so we wouldn't like to process all 
of them to paraffin either.

With best regards,
Mikael Niku
University of Helsinki, Finland

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http://lists.utsouthwestern.edu/mailman/listinfo/histonet

------------------------------

Message: 5
Date: Sun, 26 Apr 2009 07:38:53 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: [Histonet] Long term storage for IHC ?
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <863080.7096.qm <@t> web65704.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hi Mikael:
Long term storage of formalin fixed tissue is usually never a good option. If you keep them in NBF the antigenic sites could be so compromised that their unmasking could prove to be unsuccessful.
In 70% EthOL they will macerate and, as you point out, could end as if they were alcohol fixed. There are really 2 options:
1- process the tissues and  save the uncut blocks, or
2- select the most interesting pieces and fix them for 48 hours to assure full fixation and after washing in PBS freeze them and keep them frozen at -80ºC. When the moment arises that you will need them, thaw and process them. I think that  you should go with cryoperservation. I am attaching an article I wrote about formalin fixation.
René J.

Dear Histonetters,

what do you think is the best way to store formalin-fixed tissues for 
immunohistochemistry, long-term (several years)?

We are currently fixing overnight, then rinsing in buffer and storing in 
70% or 100% ethanol, +4C or -20C. However, I don't really KNOW what 
happens here and how the tissues will work after the years. I guess 
basically the formalin fixation will be at least somewhat reversed and 
after some time the tissues will be more like ethanol fixed. But are 
there better options? Long term storage in formalin will make IHC 
difficult. And this is about large numbers of tissue samples, only some 
of which will be actually used later, so we wouldn't like to process all 
of them to paraffin either.

With best regards,
Mikael Niku
University of Helsinki, Finland

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End of Histonet Digest, Vol 65, Issue 44
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