Re: VIP infiltration of rat brains

From:"John C. Dennis"

Glauco

I've had that problem, too.  I remedied it by running the three xylene
treatments for:  15'; 30'; and 30';
then paraffin for

15'; 20'; and 20'.

These are hemisected rat brains cut sagittally along the midline.

I shortened the times thinking that the real time in each solution is
longer than the time you tell the machine since filling and emptying the
retort is not instantaneous.

Hope this helps.

John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL  36849


On Wed, 25 Jul 2001, Tarozzo, Glauco wrote:

> Hi,
> I'm having  troubles in sectioning paraffin embedded rat brains and spinal
> cords that have been processed with a VIP infiltrator (Tissue-Tek VIP-E150
> Sakura). The sections brittle, tear and most notably paraffin comes apart
> from the tissue during a brief incubation in a waterbath set at 40C, before
> collecting the sections on the glass-slide. The tissues are taken from rats
> intracardially perfused  with either 4% paraformaldehyde or PLP fixative.
> Tissues are postfixed for 4 hrs at room temp., rinsed, immersed in Ethanol
> 70% o/n and subsequently processed in the VIP infiltrator. The infiltration
> cycle is the following:
>
> At 37C
> ethanol 95% 1:15'
> ethanol 95% 1:45'
> ethanol 100% 45'
> ethanol 100% 1:15'
> ethanol 100% 1:45'
> ethanol 100% 2:15'
> Xylene 1h
> Xylene 1:30'
>
> At 60C
> paraffin 1h
> paraffin 1:30
> paraffin 2:00
> paraffin 2:30
>
> I previously used this cycle on brains fixed by immersion in Carnoy fixative
> (chloroform, ethanol, acetic acid); sectioning was very easy and
> straightforward and morphology just great. Since the "symptoms" I am
> experiencing on the perfused tissues are typical of overprocessing, I tryed
> to reduce the length of xyene incubation (30' and 1h), but the sectioning
> problems remained and even worsened.
> Can anyone suggest me how to modify the conditions I'm using in order to
> overcome the troubles I mentioned?
> Thanks
>
> Glauco
>
> ___________________________________________________
> Glauco Tarozzo, PhD
> Schering-Plough Res. Inst.
> San Raffaele Science Park
> Via Olgettina, 58
> 20132 Milan - Italy
> Tel.: +39 02 2121 9222
> Fax: +39 02 2121 9242
>
>
>
>
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