[Histonet] Long term storage for IHC ?
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 ___ 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
[Histonet] help
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? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu on behalf of histonet-requ...@lists.utsouthwestern.edu Sent: Sun 4/26/2009 12:05 PM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 65, Issue 44 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... 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 mshaef...@cox.net Subject: [Histonet] paraffin embedding To: histonet@lists.utsouthwestern.edu Message-ID: b7392e4768d3431d997fb5694d0c7...@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.n...@helsinki.fi Subject: [Histonet] Long term storage for IHC? To: histonet@lists.utsouthwestern.edu Message-ID: 49f37198.5030...@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 ti...@foxmail.com Subject: Re: [Histonet] Long term storage for IHC? To: Mikael Niku mikael.n...@helsinki.fi, histonet histonet@lists.utsouthwestern.edu Message-ID: 200904261319483656...@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'件人ï¼s Mikael Niku å'é?æ-¶é-´ï¼s 2009-04-26 04:31:05 æ¶ä»¶äººï¼s histonet æSé?ï¼s 主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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Message: 4 Date: Sun, 26 Apr 2009 12:46:50 +0200 From: Gudrun Lang gu.l...@gmx.at Subject: AW: [Histonet] Long term storage for IHC? To: 'Mikael Niku' mikael.n...@helsinki.fi Cc: histonet@lists.utsouthwestern.edu Message-ID: 7224dde4d9664677a8281eecfddd9...@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
Re: [Histonet] help
Your advise to her is correct: probably the sections are not destained completely. Any section stained with the Ziehl-Nielsen Carbol Fucsin has to be treated with acid alcohol until no more red color leaches from the section no matter how much time that takes. No more red coming out of the section is the end point of the destaining. René J. --- On Sun, 4/26/09, Dertien, Janet janet.dert...@ttuhsc.edu wrote: From: Dertien, Janet janet.dert...@ttuhsc.edu Subject: [Histonet] help To: histonet@lists.utsouthwestern.edu Date: Sunday, April 26, 2009, 3:16 PM 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? -Original Message- From: histonet-boun...@lists.utsouthwestern.edu on behalf of histonet-requ...@lists.utsouthwestern.edu Sent: Sun 4/26/2009 12:05 PM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 65, Issue 44 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... 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 mshaef...@cox.net Subject: [Histonet] paraffin embedding To: histonet@lists.utsouthwestern.edu Message-ID: b7392e4768d3431d997fb5694d0c7...@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.n...@helsinki.fi Subject: [Histonet] Long term storage for IHC? To: histonet@lists.utsouthwestern.edu Message-ID: 49f37198.5030...@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 ti...@foxmail.com Subject: Re: [Histonet] Long term storage for IHC? To: Mikael Niku mikael.n...@helsinki.fi, histonet histonet@lists.utsouthwestern.edu Message-ID: 200904261319483656...@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�'件人ï¼s Mikael Niku å�'é?�æ-¶é-´ï¼s 2009-04-26 04:31:05 æ¶ä»¶äººï¼s histonet æSé?�ï¼s 主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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu
[Histonet] RE Finding Iron in Decalcified Sections
Thanks to all who have offered me advice and ideas. I've taken it on board and I'll try a few new approaches in the coming weeks. I'll let you know the outcome. Cheers, Craig Craig (Joe) Farish Senior Technical Officer Veterinary Diagnostics School of Animal and Veterinary Sciences Charles Sturt University Wagga Wagga, Australia c far...@csu.edu.au ' I don't want to achieve immortality through my work, I want to achieve immortality through not dying' - Woody Allen ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Doublecortin, Ki67, Caspase-3: need antigen retrieval ?
Hi all i am working on brain. After perfusion with 4% PFA, I put the brain in the fixative for post-fixation overnight. Then I put the brain into 30% surcose until sink. Then I perform cryostat or microtome sections. Do you think it is necessary to perform antigen retrieval of DCX (doublecortin), ki67, and caspase-3? I got very weak staining signals. I am not sure whether it is due to the low primary incubation concentration? Or insufficient antigen binding site exposure? 2009-04-27 TF ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
