RE: [Histonet] Formalin-fixed paraffin embedded question
I agree with all the comments so far, and I think all the steps are far too long, particularly the overnight in molten wax. You are effectively cooking your samples. I would avoid overnight in 100% ethanol too; it will tend to harden the tissue. Get hold of the manual for animal tissues available from the Society for histotechnology. I found this invaluable and now most of my tissues can be sectioned without soaking at all. I just chill them slightly, if necessary. Good luck with your samples. Margaret Miss Margaret Blount Histology Manager Metabolic Research Laboratories Level 4 Institute of Metabolic Science Box 289, Addenbrooke's Hospital Hills Road, Cambridge, CB2 0QQ Tel 01223 769061/336079 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Noel Gray Sent: 21 February 2011 20:55 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin-fixed paraffin embedded question I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
The NSH Manual needs to be seriously updated. There is no protocol for mouse brain that could help this guy with his problem. Andi On Feb 22, 2011, at 2:02 AM, Margaret Blount wrote: I agree with all the comments so far, and I think all the steps are far too long, particularly the overnight in molten wax. You are effectively cooking your samples. I would avoid overnight in 100% ethanol too; it will tend to harden the tissue. Get hold of the manual for animal tissues available from the Society for histotechnology. I found this invaluable and now most of my tissues can be sectioned without soaking at all. I just chill them slightly, if necessary. Good luck with your samples. Margaret Miss Margaret Blount Histology Manager Metabolic Research Laboratories Level 4 Institute of Metabolic Science Box 289, Addenbrooke's Hospital Hills Road, Cambridge, CB2 0QQ Tel 01223 769061/336079 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Noel Gray Sent: 21 February 2011 20:55 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin-fixed paraffin embedded question I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
Just got to thinking...I do animal tissue processing. I do use an automated processor, but it is open with no pressure or vacuum. If you think this will help I can give you the times I use. It is an overnight process and takes more than a work day worth of hours. Let me know? Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Margaret Blount Sent: Tuesday, February 22, 2011 3:02 AM To: Noel Gray; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Formalin-fixed paraffin embedded question I agree with all the comments so far, and I think all the steps are far too long, particularly the overnight in molten wax. You are effectively cooking your samples. I would avoid overnight in 100% ethanol too; it will tend to harden the tissue. Get hold of the manual for animal tissues available from the Society for histotechnology. I found this invaluable and now most of my tissues can be sectioned without soaking at all. I just chill them slightly, if necessary. Good luck with your samples. Margaret Miss Margaret Blount Histology Manager Metabolic Research Laboratories Level 4 Institute of Metabolic Science Box 289, Addenbrooke's Hospital Hills Road, Cambridge, CB2 0QQ Tel 01223 769061/336079 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Noel Gray Sent: 21 February 2011 20:55 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin-fixed paraffin embedded question I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
Are you volunteering? That is so nice of you =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Grantham, Andrea L - (algranth) Sent: Tuesday, February 22, 2011 9:48 AM To: Margaret Blount Cc: Noel Gray; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Formalin-fixed paraffin embedded question The NSH Manual needs to be seriously updated. There is no protocol for mouse brain that could help this guy with his problem. Andi On Feb 22, 2011, at 2:02 AM, Margaret Blount wrote: I agree with all the comments so far, and I think all the steps are far too long, particularly the overnight in molten wax. You are effectively cooking your samples. I would avoid overnight in 100% ethanol too; it will tend to harden the tissue. Get hold of the manual for animal tissues available from the Society for histotechnology. I found this invaluable and now most of my tissues can be sectioned without soaking at all. I just chill them slightly, if necessary. Good luck with your samples. Margaret Miss Margaret Blount Histology Manager Metabolic Research Laboratories Level 4 Institute of Metabolic Science Box 289, Addenbrooke's Hospital Hills Road, Cambridge, CB2 0QQ Tel 01223 769061/336079 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Noel Gray Sent: 21 February 2011 20:55 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin-fixed paraffin embedded question I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
I'd be happy to help. Maybe you can too! 8-) On Feb 22, 2011, at 8:53 AM, sgoe...@mirnarx.com wrote: Are you volunteering? That is so nice of you =) Sarah Goebel, BA, HT(ASCP) Histotechnologist Mirna Therapeutics 2150 Woodward Street Suite 100 Austin, Texas 78744 (512)901-0900 ext. 6912 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Grantham, Andrea L - (algranth) Sent: Tuesday, February 22, 2011 9:48 AM To: Margaret Blount Cc: Noel Gray; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Formalin-fixed paraffin embedded question The NSH Manual needs to be seriously updated. There is no protocol for mouse brain that could help this guy with his problem. Andi On Feb 22, 2011, at 2:02 AM, Margaret Blount wrote: I agree with all the comments so far, and I think all the steps are far too long, particularly the overnight in molten wax. You are effectively cooking your samples. I would avoid overnight in 100% ethanol too; it will tend to harden the tissue. Get hold of the manual for animal tissues available from the Society for histotechnology. I found this invaluable and now most of my tissues can be sectioned without soaking at all. I just chill them slightly, if necessary. Good luck with your samples. Margaret Miss Margaret Blount Histology Manager Metabolic Research Laboratories Level 4 Institute of Metabolic Science Box 289, Addenbrooke's Hospital Hills Road, Cambridge, CB2 0QQ Tel 01223 769061/336079 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Noel Gray Sent: 21 February 2011 20:55 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Formalin-fixed paraffin embedded question I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
According with your protocol it seems that you are processing by hand. Even so, the periods seem too long, specially for brain using xylene. The best (and final) solution for your problem will be to abandon xylene and start using mineral oil. René J. --- On Mon, 2/21/11, Noel Gray gr...@upstate.edu wrote: From: Noel Gray gr...@upstate.edu Subject: [Histonet] Formalin-fixed paraffin embedded question To: histonet@lists.utsouthwestern.edu Date: Monday, February 21, 2011, 3:54 PM I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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] Formalin-fixed paraffin embedded question
Noel, I agree. Your tissue isn't fixed well enough and the dehydration steps seem long, especially since at some point you split the brain and the spinal cord. After perfusion immerse in 10% NBF (a few changes) at least 24 hrs (I like to leave it in formalin longer). If you are fixing the brain whole remember that formalin is a slow fixative and it takes some time to penetrate all the way into the brain even if it was perfused first. I found that by combining perfusion with immersion in the fixative the brain fixed and cut better. Before you start dehydrating, try washing the tissue in running water at least an hour (or longer). Then start your dehydration. Xylene makes tissue brittle and overnite in xylene I think isn't doing you any favors. I use Clear Rite 3 for my clearing agent. Are you hand processing? Do do have access to vacuum infiltration? I have a protocol for mouse brains but it is for processing on a VIP. When cutting try soaking the block at room temp in water to which a bit of glycerin has been added. I usually don't place the brains on ice but just in cool water and my room is always cold. I read here on histonet that people let their tissues soak for a few minutes but with mouse tissue you need to soak it longer. I don't really time the soaking but it is way longer than a few minutes. Then you might try wiping the surface of the block with a gauze sponge or even a cotton tipped applicator that was dipped in the soaking water in between sections as you are cutting your ribbon - don't make it too wet just moist. Andi On Feb 21, 2011, at 1:54 PM, Noel Gray wrote: I am having an issue with formalin-fixed, paraffin embedded tissue that I am sectioning. I am using a microtome to cut the tissue (mouse brain, cerebellum and spinal cord) in 10 um sections which I will stain using cressyl violet. Sometimes, the tissue in a block will splinter once it hits the blade. Usually all samples from the same animal splinter but this is not always the case. If I put the block into the water bath (sectioning surface exposed to water or not) this seems to stop the splintering for 1-200 um worth of tissue. However, I am afraid this may bring error into the histological analysis of my tissue. I assume it has something to do with the protocol I am using to prepare the tissue. I guessed that maybe the dehydration, alcohol clearing, or paraffin infiltration are not complete, resulting in the problem I have. However, I looked at various FFPE protocols and each of my wash steps are longer, which may be the problem? I was wondering if anyone has encountered this before, or if anyone knows exactly what is going on with my tissue and how I can fix it? Thank you. Here is my protocol: -Anesthetize mouse followed by a system flush of 30 ml of PBS and then slow perfusion of 50 ml of 4% PFA. -Brain and spinal cord are removed as a single, in tact unit and placed into 70% ethanol for 4 hours -80% EtOH for 4 hours -90% EtOH over night -100% EtOH #1 for 4 hours -100% EtOH #2 for 4 hours -100% EtOH #3 over night, forebrain cerebellum and spinal cord are separated -Xylene wash #1 for 4 hours -Xylene wash #2 for 4 hours -Xylene wash #3 over night -Moulton paraffin wash #1 for 4 hours -Moulton paraffin wash #2 for 4 hours -Moulton paraffin wash #3 over night -Tissue is embedded and then sectioned into 10 um sections Again thank you for your time. Noel W Gray Neuroscience Graduate Program SUNY Upstate Medical University 3219 Weiskotten Hall 766 Irving Ave Syracuse, NY 13210-1630 (315) 464-8144 gr...@upstate.edu ___ 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
