RE: FW: brain section blowout
|From:||"Rich, Nina J Ms WACH" <Nina.Rich@se.amedd.army.mil>|
I personally have not worked with mouse brain per se but I can give you some
pointer on brain in gereral.
Fixation is very important but not the only cause of tissue splitting up.
Brain is very sensitive to heat
so whenever you cut it the water bath needs to be turned down it will seem
luke warm instead of hot. Also use plus slides when you pick them up. Once
you have gotten a good section by turning the water bath down don't stain
them until the next day. The key is to let them dry on the slides
overnight. This seems to adherr them better and they don't wash off. You
may have an occassional one to wash but just recut it and let it air dry
longer and it should be ok. I guess you might have to play around with your
slides and see how long they need to sit because they are mouse brain
instead of human. I would think they might not have to sit as long since it
is smaller sections. It does not work as well if you leave them in a dryer
longer either. It has to be air-dryed. Hope this works for you. Jean Rich
From: Anthony f. Brandwood [mailto:email@example.com]
Sent: Tuesday, May 29, 2001 5:26 AM
To: J. A. Kiernan; Stephanie Rodriguez
Subject: Re: FW: brain section blowout
--On 28 May 2001 10:39 -0400 "J. A. Kiernan" <firstname.lastname@example.org> wrote:
> 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
We have also had this problem occuring from time to time with formalin
fixation but it has always happened to one or two blocks of CNS tissue
which have been processed with other CNS blocks and these have cut without
any problem at all. The blocks had not been jammed tight in the basket
during processing and all the solvents had been reasonably fresh and the
processing schedule been used for many years to process CNS tissue without
many problems. Any Suggestions?
Department of Veterinary Pathology,
University of Liverpool,
Tel. No. 0151-794 4251
Fax. No. 0151-794 4268
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