[Histonet] problems with mouse brain fixation

[Martina Urbanek]

Hello everybody on histonet,

we have some problems with handling mouse (21 and 90 days old) and rat brains.
We do mouse brain perfusion using 4% paraformaldehyde in PBS, pH 7.4. We use
gravity for perfusion for about 30 minutes (volume about 80 ml) and after
perfusion we leave the brains over night in the same fixative. Then we process
in a Shandon tissue processor (70% Alcohol, 80% Alcohol, 95% Alcohol, 3 changes
100% alcohol, 3 changes xylene, 2 changes paraffin 56°C; time depends on size
of tissue). Now we have the problem that some brains are too hard and some also
seem to shrink more than others, when we cut them and put them on waterbath
they seem to expand and distort and often brittle. So that it looks like only
fibrous tissue is left, the structure is gone. I already had a look on histonet
archive but did not find anything that helps, therefore I hope that someone has
an idea what can be wrong. I have to say that we don´t have any problems when
we immersion fix the brains with 4% formaldehyde.
The problems we have with rat brains are a bit different, because we get the
brains from another group who perfuse the lung with 4% paraformaldehyde in
Hepes-buffer, pH 7.35. We then postfix over night in the same fixative they
use (when the brain is also perfused) or for 3 days (when brain is not
perfused). After dehydration in tissue processor we have the same cutting
problems like we observe with mouse brain tissue, even worse.
Any suggestions are appreciated!

Thank you very much for your help!!!

Martina Urbanek



Ms. Martina Urbanek
Forschungslabor der
Klin.Abt. für Neonatologie
neonatal neuroscience research laboratory
Med. University Innsbruck
Innrain 66, 4th floor
A-6020 Innsbruck
Tel. +43 (0)512 504 27755/27765
Fax: +43 (0)512 504 27766
Email: Martina.Urbanek <@t> uibk.ac.at