Re: RE: Re: [Histonet] 70% alcohol fixation of brain
I did try the frozen section from fresh tissue...and fix them in cold acetone for 10 min, transfer to cold PBS for 30 min, then wash in PBS at RT, begin IHC. But the tissue quality is really to bad. You always saw pieces of tissues floating, fall in to small pieces and gone when you washing the slide... Under the microscope...the background is high, and you can only see granule-like existences filled by small cavities. Actually I frozed the OCT-embedded tissue with liquid nitrogen..dont know why the tissue quality is still so bad! 2009-05-17 TF 发件人： Patsy Ruegg 发送时间： 2009-05-17 01:19:09 收件人： ti...@foxmail.com; 'Geoff McAuliffe' 抄送： Histonet@lists.utsouthwestern.edu 主题： RE: Re: [Histonet] 70% alcohol fixation of brain U would be better off freezing the brain unfixed than trying to fix in alcohol and freeze it, alcohol fixation is not recommended for freezing tissue. Just freeze slices of the brain or a whole rat or mouse brain and then make sections and fix the sections in alcohol/acetone mixture. Sectioning is not easy but this is a better approach than fixing in alcohol and then trying to freeze. I would try to get some frozen sections from the unfixed frozen brain for use with antibodies not formalin friendly and then thaw fix the frozen block in formalin for several day, infiltrate it in 30% sucrose and refreeze, you should be able to get better sections then, but the tissue has to be fixed before you can cryoprotect in sucrose. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech, LLC Fitzsimmons BioScience Park 12635 Montview Blvd. Suite 215 Aurora, CO 80010 P-720-859-4060 F-720-859-4110 email pru...@ihctech.net website www.ihctech.net IHC Resource Group www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of TF Sent: Friday, May 15, 2009 12:15 PM To: Geoff McAuliffe Cc: Histonet@lists.utsouthwestern.edu Subject: Re: Re: [Histonet] 70% alcohol fixation of brain thanks to the reply. some antigens will be damaged by PFA fixation and can not be retreieval. the shock frozen section with acetone is of quite bad tissue quality. therefore I am trying alcohol! 2009-05-16 TF 发件人： Geoff McAuliffe 发送时间： 2009-05-15 23:55:15 收件人： tifei 抄送： Histonet@lists.utsouthwestern.edu 主题： Re: [Histonet] 70% alcohol fixation of brain Greetings TF: Good luck freezing a brain that is full of alcohol! Have you checked the freezing point of alcohol? Why are you doing this? Immersing a whole brain in 70% alcohol, why?? 30% sucrose is a cryoprotectant so the brain is not full of holes from ice crystals.Alcohol defeats this purpose. Geoff TF wrote: > Hi, i dont want to use PFA for the brain fixation (rat). > Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I > do post-fixation at room temperature for 24 hours. > I also tried saline perfusion, then I directly put the whole brain into 70% > alcohol. > Is this fine? > > Also, for dehydration before cutting frozen sections on a cryostat/microtome, > should I use 95% alcohol? I am now using 30% sucrose~ > > 2009-05-15 > > > > TF > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: Re: [Histonet] 70% alcohol fixation of brain
U would be better off freezing the brain unfixed than trying to fix in alcohol and freeze it, alcohol fixation is not recommended for freezing tissue. Just freeze slices of the brain or a whole rat or mouse brain and then make sections and fix the sections in alcohol/acetone mixture. Sectioning is not easy but this is a better approach than fixing in alcohol and then trying to freeze. I would try to get some frozen sections from the unfixed frozen brain for use with antibodies not formalin friendly and then thaw fix the frozen block in formalin for several day, infiltrate it in 30% sucrose and refreeze, you should be able to get better sections then, but the tissue has to be fixed before you can cryoprotect in sucrose. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech, LLC Fitzsimmons BioScience Park 12635 Montview Blvd. Suite 215 Aurora, CO 80010 P-720-859-4060 F-720-859-4110 email pru...@ihctech.net website www.ihctech.net IHC Resource Group www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of TF Sent: Friday, May 15, 2009 12:15 PM To: Geoff McAuliffe Cc: Histonet@lists.utsouthwestern.edu Subject: Re: Re: [Histonet] 70% alcohol fixation of brain thanks to the reply. some antigens will be damaged by PFA fixation and can not be retreieval. the shock frozen section with acetone is of quite bad tissue quality. therefore I am trying alcohol! 2009-05-16 TF 发件人： Geoff McAuliffe 发送时间： 2009-05-15 23:55:15 收件人： tifei 抄送： Histonet@lists.utsouthwestern.edu 主题： Re: [Histonet] 70% alcohol fixation of brain Greetings TF: Good luck freezing a brain that is full of alcohol! Have you checked the freezing point of alcohol? Why are you doing this? Immersing a whole brain in 70% alcohol, why?? 30% sucrose is a cryoprotectant so the brain is not full of holes from ice crystals.Alcohol defeats this purpose. Geoff TF wrote: > Hi, i dont want to use PFA for the brain fixation (rat). > Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I > do post-fixation at room temperature for 24 hours. > I also tried saline perfusion, then I directly put the whole brain into 70% > alcohol. > Is this fine? > > Also, for dehydration before cutting frozen sections on a cryostat/microtome, > should I use 95% alcohol? I am now using 30% sucrose~ > > 2009-05-15 > > > > TF > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: Re: [Histonet] 70% alcohol fixation of brain
thanks to the reply. some antigens will be damaged by PFA fixation and can not be retreieval. the shock frozen section with acetone is of quite bad tissue quality. therefore I am trying alcohol! 2009-05-16 TF 发件人： Geoff McAuliffe 发送时间： 2009-05-15 23:55:15 收件人： tifei 抄送： Histonet@lists.utsouthwestern.edu 主题： Re: [Histonet] 70% alcohol fixation of brain Greetings TF: Good luck freezing a brain that is full of alcohol! Have you checked the freezing point of alcohol? Why are you doing this? Immersing a whole brain in 70% alcohol, why?? 30% sucrose is a cryoprotectant so the brain is not full of holes from ice crystals.Alcohol defeats this purpose. Geoff TF wrote: > Hi, i dont want to use PFA for the brain fixation (rat). > Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I > do post-fixation at room temperature for 24 hours. > I also tried saline perfusion, then I directly put the whole brain into 70% > alcohol. > Is this fine? > > Also, for dehydration before cutting frozen sections on a cryostat/microtome, > should I use 95% alcohol? I am now using 30% sucrose~ > > 2009-05-15 > > > > TF > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] 70% alcohol fixation of brain
> Good luck freezing a brain that is full of alcohol! Have you checked the freezing point of alcohol? > Why are you doing this? I think that moire often than not, people ask before they think ;-) On Fri, May 15, 2009 at 11:56 AM, Geoff McAuliffe wrote: > Greetings TF: > > Good luck freezing a brain that is full of alcohol! Have you checked the > freezing point of alcohol? > Why are you doing this? > Immersing a whole brain in 70% alcohol, why?? > 30% sucrose is a cryoprotectant so the brain is not full of holes from ice > crystals.Alcohol defeats this purpose. > > Geoff > > > > TF wrote: > >> Hi, i dont want to use PFA for the brain fixation (rat). >> Now I tried to perfuse the rat with saline, followed with 70% alcohol. >> Then I do post-fixation at room temperature for 24 hours. >> I also tried saline perfusion, then I directly put the whole brain into >> 70% alcohol. >> Is this fine? >> >> Also, for dehydration before cutting frozen sections on a >> cryostat/microtome, should I use 95% alcohol? I am now using 30% sucrose~ >> >> 2009-05-15 >> >> >> TF ___ >> Histonet mailing list >> Histonet@lists.utsouthwestern.edu >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet >> >> >> >> > > > -- > -- > ** > Geoff McAuliffe, Ph.D. > Neuroscience and Cell Biology > Robert Wood Johnson Medical School > 675 Hoes Lane, Piscataway, NJ 08854 > voice: (732)-235-4583 mcaul...@umdnj.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] 70% alcohol fixation of brain
Greetings TF: Good luck freezing a brain that is full of alcohol! Have you checked the freezing point of alcohol? Why are you doing this? Immersing a whole brain in 70% alcohol, why?? 30% sucrose is a cryoprotectant so the brain is not full of holes from ice crystals.Alcohol defeats this purpose. Geoff TF wrote: Hi, i dont want to use PFA for the brain fixation (rat). Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I do post-fixation at room temperature for 24 hours. I also tried saline perfusion, then I directly put the whole brain into 70% alcohol. Is this fine? Also, for dehydration before cutting frozen sections on a cryostat/microtome, should I use 95% alcohol? I am now using 30% sucrose~ 2009-05-15 TF ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- -- ** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583 mcaul...@umdnj.edu ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] 70% alcohol fixation of brain
Hi, i dont want to use PFA for the brain fixation (rat). Now I tried to perfuse the rat with saline, followed with 70% alcohol. Then I do post-fixation at room temperature for 24 hours. I also tried saline perfusion, then I directly put the whole brain into 70% alcohol. Is this fine? Also, for dehydration before cutting frozen sections on a cryostat/microtome, should I use 95% alcohol? I am now using 30% sucrose~ 2009-05-15 TF ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
