Re: VIP infiltration of rat brains
Wow! I would have to say that I consider your whole
program way too harsh for rat tissue. The schedule we
use has no steps longer than 1 hr except for the last
xylene, and paraffin steps are all 1 hr. We have
incorporated the usual precautions of checking the 1st
paraffin (at least) before processing (if it's "oily"
feeling, we change that paraffin and rotate the others
down 1 stage). The other thing that you might check
is the paraffin itself. We normally section at 45C,
but one whole lot ended up requiring lower temps in
the water baths, and even then had problems with
tissue/paraffin separation, exploding sections, the
whole bit. We have used this processing schedule for
both routine histology and immuno (abbreviated
fixation). Since Murphy's Law hasn't been repealed,
we still have the occasional hiccup, but overall our
processing gives us excellent results.
--- "Tarozzo, Glauco"
> 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 40#176#C, 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 37#176#C
> 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 60#176#C
> 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?
> 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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