Methylenblue doesn't adhere to the tissue properly, if the acid isn't washed out well with tapwater. The staining pH has to be alkalineto make a overall stain. Methylenblue is washed out easily in diluted ethanols. Just wash in tapwater, blot on paper and let dry for a few seconds and dehydrate quickly in absolute ethanol to xylen (or similar).
Some tissue (especially connective tissue) tends to refuse methylenblue staining. Gudrun Lang -----Ursprüngliche Nachricht----- Von: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Dertien, Janet Gesendet: Sonntag, 26. April 2009 21:17 An: histonet@lists.utsouthwestern.edu Betreff: [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 æS"é?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 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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Mikael Niku Gesendet: Samstag, 25. April 2009 22:25 An: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ------------------------------ Message: 5 Date: Sun, 26 Apr 2009 07:38:53 -0700 (PDT) From: Rene J Buesa <rjbu...@yahoo.com> Subject: [Histonet] Long term storage for IHC ? To: histonet@lists.utsouthwestern.edu Message-ID: <863080.7096...@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 _______________________________________________ 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 End of Histonet Digest, Vol 65, Issue 44 **************************************** _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet