TUNEL in brain slices
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Dear Confocalists, Does anybody have a good protocol for performing TUNEL on Paraformaldehyde fixed brain slices? I am having an awful time doing it in flash-frozen fresh brain slices, mainly because my brains keep cracking when I cut them in the cryostat. I have tried everything: playing with the temperature range, dulling the knife blade, lighting candles to the gods of the cryostat..... I am thinking that PF would take care of the water in the brains (which I think is crystallizing and cracking the brains.....), but I have never done TUNEL on fixed slices..... Please help! Gil Palchik |
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Do you put your brains in a sucrose gradient before freezing for cryosections??? Cracking should not occur if you do this.
-----Original Message----- From: Guillermo Palchik <[hidden email]> Date: Tue, 24 Jun 2008 11:05:50 To:[hidden email] Subject: TUNEL in brain slices Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Confocalists, Does anybody have a good protocol for performing TUNEL on Paraformaldehyde fixed brain slices? I am having an awful time doing it in flash-frozen fresh brain slices, mainly because my brains keep cracking when I cut them in the cryostat. I have tried everything: playing with the temperature range, dulling the knife blade, lighting candles to the gods of the cryostat..... I am thinking that PF would take care of the water in the brains (which I think is crystallizing and cracking the brains.....), but I have never done TUNEL on fixed slices..... Please help! Gil Palchik ************ There are people that fight one day and are good... There are those that fight one year and are better... There are people that fight many years and are better still... But there are those who fight their entire lives: those are indispensable... Bertolt Brecht (1898-1956) How can it be that mathematics, being after all a product of human thought which is independent of experience, is so admirably appropriate to the objects of reality? Is human reason, then, without experience, merely by taking thought, able to fathom the properties of real things? Albert Einstein (1879-1955) |
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In reply to this post by Guillermo Palchik
Search the CONFOCAL archive at
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Used to cryostat unfixed mouse cerebellum, I just immersed it in OCT or tissue TEK,
in a plastic bijou, and froze it in dry ice. Broke the plastic bijou off the block with
pliers (goggles would be useful) and attached it to the chuck with more OCT. Carve
off the excess OCT and it worked fine for me. Peter From: Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear
Confocalists, Does
anybody have a good protocol for performing TUNEL on Paraformaldehyde fixed
brain slices? I am having an awful time doing it in flash-frozen fresh brain
slices, mainly because my brains keep cracking when I cut them in the cryostat.
I have tried everything: playing with the temperature range, dulling the knife
blade, lighting candles to the gods of the cryostat..... I am thinking that PF
would take care of the water in the brains (which I think is crystallizing and
cracking the brains.....), but I have never done TUNEL on fixed slices..... Please
help! Gil
Palchik |
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In reply to this post by Guillermo Palchik
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Andrea,
Currently, I am flash-freezing the brains by scooping them out of the skull and placing them on cold (~-50 C) isopentane for a less than a minute. After that, I let them dry on dry ice for a minute or so, and then I wrap them in tinfoil and they go into the -80 C freezer until it is time to cut them. When I go to cut them, i let them equilibrate in the cryostat overnight, to the desired temperature (~-20 C), and then I cut them (or try to cut them...). The brains are not fixed, so I don't know if placing them in the sucrose for cryoprotection would work.... Gil
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Search the CONFOCAL archive at
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Ah,my mistake, I thought you were fixing them first.
For our fresh frozen brains, and we get nice morphology for
immunostains and bet-galactosidase staining, we place freshly
extracted mouse brains into OCT and freeze by floating on a bath of
liquid nitrogen in a metal tray. We get no artifacts in the sections.
For work which requires very high morphological criterion and for
thick floating sections we perfuse the animals.
However, I have done TUNEL on paraffin sections and use the kit
from Roche or Promega, both work well in my experience.
Hi Andrea, Currently, I am flash-freezing the brains by scooping them out of the skull and placing them on cold (~-50 C) isopentane for a less than a minute. After that, I let them dry on dry ice for a minute or so, and then I wrap them in tinfoil and they go into the -80 C freezer until it is time to cut them. When I go to cut them, i let them equilibrate in the cryostat overnight, to the desired temperature (~-20 C), and then I cut them (or try to cut them...). The brains are not fixed, so I don't know if placing them in the sucrose for cryoprotection would work.... Gil Subject:Re: TUNEL in brain slicesFrom:"Andrea T. Hooper" <[hidden email]>Reply-To:[hidden email]Date:Tue, 24 Jun 2008 15:18:53 +0000Content-Type:text/plain Do you put your brains in a sucrose gradient before freezing for cryosections??? Cracking should not occur if you do this. To:[hidden email] -- |
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In reply to this post by Guillermo Palchik
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hello Guillermo, I make a protocol of TUNEL on
fixed brain slices with the kit of PROMEGA and the results are very good. The
protocol is expalined in the kit but I can to send
you.
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal HI, I agree with him.The kit which we use from Promega is 'DeadEnd™ Fluorometric TUNEL System'.We are using paraffin embedded section. Regards, Vaishali --- On Wed, 6/25/08, Sagrario Callejo <[hidden email]> wrote: > From: Sagrario Callejo <[hidden email]> > Subject: Re: TUNEL in brain slices > To: [hidden email] > Date: Wednesday, June 25, 2008, 1:07 PM > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello Guillermo, I make a protocol of TUNEL on fixed brain > slices with the kit of PROMEGA and the results are very > good. The protocol is expalined in the kit but I can to > send you. |
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