Re: FW: brain section blowout
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Fri, 25 May 2001, Stephanie Rodriguez wrote:
> Date: Fri, 25 May 2001 08:13:15 -0700
> When I float my ribbons out on my water bath, the sections rapidly come
> apart before I have a chance to get them on a slide.
> Could it be that the tissue is not adequately fixed? or perhaps poorly
It has to be one or the other or both. For reasons given below
I think your difficulties are due to improper processing after
> I fixed the whole mouse brains in 10% NBF for 8hrs at RT, then
> 72hrs at 4C, then sent them to the contract lab ...
The 3 days at 4C is a bit strange. If you want minimal formaldehyde
fixation, why not move the specimens into 70% alcohol after a few
hrs (and thereby obtain mostly alcohol fixation)? If you want real
formaldehyde fixation, why do it in a cold place that can only slow
down the chemical reactions?
Formaldehyde isn't a _very_ good fixative when you want paraffin
sections, but it is _fairly_ good, and it's also routinely used
by nearly everyone, so its bad effects (differential shrinkage,
weary-looking nuclei etc) are so widely recognized that they
are passed off as "normal."
Fragmentation of paraffin-embedded tissue when floated on a
warm water bath is _not_ one of the faults of formaldehyde or
any other fixative. Your problem has to be the fault of the
contract lab that collects money for not dehydrating, clearing,
infiltrating and properly blocking out your specimens.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
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