embedding medium
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Hi,
Does anyone know of an embedding medium which can be used in place of paraffin, to reduce the autofluoresence produced? Is there any literature available related to this topic? Thanks. |
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Howdy,
You could try polyester wax. It is a bit trickier to cut than paraffin since it has to be kept cold otherwise it will be too soft to cut. you will also need to float the sections onto ice cold water instead of warm. In my experience i have not seen autofluorecnce due to paraffin wax, usually it is due to the tissue type used. Cheers Cam Cameron J. Nowell Microscpy Manager Central Resource for Advanced Microscopy Ludwig Insttue for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 http://www.ludwig.edu.au/branch/research/platform/microscopy.htm ________________________________ From: Confocal Microscopy List on behalf of samrina aslam Sent: Thu 16/04/2009 6:30 PM To: [hidden email] Subject: embedding medium Hi, Does anyone know of an embedding medium which can be used in place of paraffin, to reduce the autofluoresence produced? Is there any literature available related to this topic? Thanks. |
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Dear Cameron,
Thank you for your message. Okay is there a good method to reduce the autofluoresence? I am looking at amyloid deposits in various organs. However the autofluoresence is the same for all of them. I have tried the pressure cooker method for antigen retrieval but that has not reduced the background at all. Thanks. Samrina > Date: Thu, 16 Apr 2009 19:20:03 +1000 > From: [hidden email] > Subject: Re: embedding medium > To: [hidden email] > > Howdy, > > You could try polyester wax. It is a bit trickier to cut than paraffin since it has to be kept cold otherwise it will be too soft to cut. you will also need to float the sections onto ice cold water instead of warm. > > In my experience i have not seen autofluorecnce due to paraffin wax, usually it is due to the tissue type used. > > Cheers > > > Cam > > > > Cameron J. Nowell > Microscpy Manager > Central Resource for Advanced Microscopy > Ludwig Insttue for Cancer Research > PO Box 2008 > Royal Melbourne Hospital > Victoria, 3050 > AUSTRALIA > > Office: +61 3 9341 3155 > Mobile: +61422882700 > Fax: +61 3 9341 3104 > > http://www.ludwig.edu.au/branch/research/platform/microscopy.htm > > > ________________________________ > > From: Confocal Microscopy List on behalf of samrina aslam > Sent: Thu 16/04/2009 6:30 PM > To: [hidden email] > Subject: embedding medium > > > > Hi, > > Does anyone know of an embedding medium which can be used in place of > paraffin, to reduce the autofluoresence produced? > > Is there any literature available related to this topic? > > Thanks. " Upgrade to Internet Explorer 8 Optimised for MSN. " Download Now |
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In reply to this post by Samrina Aslam
samrina aslam wrote:
> Does anyone know of an embedding medium which can be used in place of > paraffin, to reduce the autofluoresence produced? > > Is there any literature available related to this topic? I'm unaware of paraffin causing autofluorescence. The usual causes in histochemistry are: 1) Glutaraldehyde fixation, which produces high levels of autofluorescence. 2) Formaldehyde fixation for extended periods of time--also can cause autofluorescence 3) Lipofuscin--an autofluorescent pigment which accumulates in lysosomes with age. It's more of a problem with older animals than younger ones. --Autofluorescence from formaldehyde or low levels of glutaraldehyde can be treated with NaBH4. See: "Reduction of background autofluorescence in brain sections following immersion in sodium borohydride." Clancy B. and Cauller LJ. J. Neuroscience Methods. 83(2):97-102, 1998 Sep 1. --Lipfuscin autofluorescence can be decreased with Cu++ ion or Sudan Black. "Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue." Schnell SA. Staines WA. Wessendorf MW. J. Histochemistry & Cytochemistry. 47(6):719-30, 1999 Jun. Good luck! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email] ** |
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In reply to this post by Samrina Aslam
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In reply to this post by Martin Wessendorf-2
Most mineral oils are fluorescent, so paraffin wax might well be. But normally it's removed. Maybe the problem is insufficient removeal of the wax before mounting?
Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf Sent: Thursday, 16 April 2009 11:16 PM To: [hidden email] Subject: Re: embedding medium samrina aslam wrote: > Does anyone know of an embedding medium which can be used in place of > paraffin, to reduce the autofluoresence produced? > > Is there any literature available related to this topic? I'm unaware of paraffin causing autofluorescence. The usual causes in histochemistry are: 1) Glutaraldehyde fixation, which produces high levels of autofluorescence. 2) Formaldehyde fixation for extended periods of time--also can cause autofluorescence 3) Lipofuscin--an autofluorescent pigment which accumulates in lysosomes with age. It's more of a problem with older animals than younger ones. --Autofluorescence from formaldehyde or low levels of glutaraldehyde can be treated with NaBH4. See: "Reduction of background autofluorescence in brain sections following immersion in sodium borohydride." Clancy B. and Cauller LJ. J. Neuroscience Methods. 83(2):97-102, 1998 Sep 1. --Lipfuscin autofluorescence can be decreased with Cu++ ion or Sudan Black. "Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue." Schnell SA. Staines WA. Wessendorf MW. J. Histochemistry & Cytochemistry. 47(6):719-30, 1999 Jun. Good luck! Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 **MY E-MAIL ADDRESS HAS CHANGED. PLEASE USE [hidden email] ** No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.59/2063 - Release Date: 16/04/2009 4:38 PM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.59/2063 - Release Date: 16/04/2009 4:38 PM |
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