[Histonet] Mice embryonic brain - EM Poor tissue quality - need help

[Geoff McAuliffe]

Hi Rowani:

    Immersing the whole brain in fixative will not allow good 
ultrastructure, no matter what the fixative is. Even immersing small 
(1mm cubed) pieces will not allow optimal ultrastructure. There is no 
advantage to using cold fixative, in fact it may make your results worse 
due to slowing of the reaction of fixative with the tissue. Finally, 
embryonic tissue is more difficult to fix (well) than adult tissue.
     I would recommend vascular perfusion with a glutaraldehyde or 
glut-paraformaldehyde fixative. Consult the literature for one or more  
exact recommendations for the part of the brain you are interested in 
and the age of the embryo. The concentration of the buffer you use and 
the presence of additives (sucrose) will be more important than the 
exact concentrations of glut. or para.

Geoff

Rowani wrote:

>Hi
>My electron microscopy sections of brain of mice foetuses (18.5 dpc)  are
>difficult to interpret because of  disrupted and lysed cells. The connective
>tissue surrounding the cells  appears disintegrated with empty
>spaces/vacuoles like artifact ?.  The brain was processed according to the
>following protocols: brain was dissected and immersed in ice cold fixative
>(glutaryldehyde 2.5%, paraformaldehyde 4%, sucrose 2% in PBS pH 7.2) for 1-2
>hrs to firm it up before cutting them to smaller fragments (2 cumm). After
>an overnight fixation, re-fixed in osmium tetroxide, washed in PBS,
>dehydrated through a series of ethanol (70%, 90%, 95% and 100%) and embedded
>in araldyte resin.  The brain of adult mice processed using similiar
>protocol was excellent.  COuld someone suggest ways to improve the tissue
>quality ?
>
>TQ
>
>Rowani
>
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-- 
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**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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