[Histonet] drosophila sectioning
Anthony Gatt
Anthony.Gatt <@t> jefferson.edu
Fri May 11 13:25:38 CDT 2007
thanks, James. Just curious, what is T&S under the agitation? I only have the options for "on" or "off" on my processor. Also do you use the same paraffin for embedding?
---- Original message ----
>Date: Fri, 11 May 2007 09:59:07 -0700
>From: "James Watson" <jwatson <@t> gnf.org>
>Subject: RE: [Histonet] drosophila sectioning
>To: <Anthony.Gatt <@t> jefferson.edu>, "Rene J Buesa" <rjbuesa <@t> yahoo.com>, <histonet <@t> lists.utsouthwestern.edu>
>
>
>
> Anthony,
>
>
>
> This is the processing schedule that I have used on
> drosophila heads, note that I use 5% Glycerin in
> the absolute alcohols. The fixation was formalin
> or 4% PFA with no softening agent used other than
> the glycerin.
>
>
>
>
>
>
>
> Drosophila Processing Schedule
>
>+--------------------------------------------------------+
>|Drosophila Tissue Processing Schedule |
>| |
>|+------------------------------------------------------+|
>||Station|Reagent |Temperature|Vacuum|Time|Drain|Agitate||
>|| | | | | |Time | ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 1 |70% ETOH| Ambient | OFF |0:15| 30 |Off ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 2 |80% ETOH| Ambient |Vacuum|0:20| 30 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 3 |95% ETOH| Ambient |Vacuum|0:15| 30 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 4 |95% ETOH| Ambient |Vacuum|0:20| 45 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 5 | 5% | Ambient |Vacuum|0:15| 30 |Stirred||
>|| |Glycerin| | | | | ||
>|| | in | | | | | ||
>|| | | | | | | ||
>|| | 100% | | | | | ||
>|| | ETOH | | | | | ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 6 | 5% | Ambient |Vacuum|0:20| 30 | T&S ||
>|| |Glycerin| | | | | ||
>|| | in | | | | | ||
>|| | | | | | | ||
>|| | 100% | | | | | ||
>|| | ETOH | | | | | ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 7 | 5% | Ambient |Vacuum|0:30| 100 | T&S ||
>|| |Glycerin| | | | | ||
>|| | in | | | | | ||
>|| | | | | | | ||
>|| | 100% | | | | | ||
>|| | ETOH | | | | | ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 8 | Xylene | Ambient |Vacuum|0:15| 45 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 9 |Xylene | Ambient |Vacuum|0:20| 45 |T&S ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 10 | Xylene | Ambient |Vacuum|0:30| 120 | T&S ||
>||-------+--------+-----------+------+----+-----+-------||
>|| 11 |Paraffin| 60 |Vacuum|0:15| 120 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 12 |Paraffin| 60 |Vacuum|0:20| 120 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 13 |Paraffin| 60 |Vacuum|0:20| 120 |Stirred||
>||-------+--------+-----------+------+----+-----+-------||
>|| 14 |Paraffin| 60 |Vacuum|0:30| 120 |Stirred||
>|+------------------------------------------------------+|
>| |
>| |
>| |
>| |
>+--------------------------------------------------------+
>
>
>
>
>
>
>
>
>
>
>
> James Watson HT ASCP
>
> Facilities Manager of Histology
>
> GNF, Genomics Institute of the Novartis Research
> foundation
>
> jwatson <@t> gnf.org
>
> 858-332-4647
>
>
>
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu]
> On Behalf Of Anthony Gatt
> Sent: Friday, May 11, 2007 9:36 AM
> To: Rene J Buesa; Anthony.Gatt <@t> jefferson.edu;
> histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] drosophila sectioning
>
>
>
> Thanks, Rene. Any advice on a softening agent and
> where it would fit into the protocol?
>
>
>
> ---- Original message ----
>
> >Date: Fri, 11 May 2007 09:15:23 -0700 (PDT)
>
> >From: Rene J Buesa <rjbuesa <@t> yahoo.com>
>
> >Subject: Re: [Histonet] (no subject)
>
> >To: Anthony.Gatt <@t> jefferson.edu,
> "histonet <@t> lists.utsouthwestern.edu"
> <histonet <@t> lists.utsouthwestern.edu>
>
> >
>
> > Your problem resides in the fact that you have
> to
>
> > "soften" the chitin before processing the
> heads,
>
> > otherwise the chitin cannot be infiltrated by
> the
>
> > paraffin, causing the tears you are having.
>
> > Soften the chitin first and process
> afterwards.
>
> > René J.
>
> >
>
> > Anthony Gatt <Anthony.Gatt <@t> jefferson.edu>
> wrote:
>
> >
>
> > Hello, I am currently sectioning
> drosophila heads
>
> > and am tearing up the paraffin. Without
> the heads,
>
> > I get beautiful ribbons so I suspect it is
> the
>
> > sample itself. I am using a tissue
> processor with
>
> > the following protocol after an o/n fix in
> 4% PFA.
>
> >
>
> > 15m -alcoholic formalin (x2)
>
> > 30m -95% EtOH (x2)
>
> > 30m -100% EtOH (x2)
>
> > 30m -Histoclear (x3)
>
> > 30m -Paraffin (x2)
>
> > 45m -Paraffin
>
> >
>
> > Embed immediately in paraffin, cold block
> 30m,
>
> > section next day.
>
> >
>
> > I am new to this an would greatly
> appreciate any
>
> > help.
>
> >
>
> > Thank you,
>
> >
>
> > Anthony
>
> >
>
> >
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