You are freezing too slowly. Cryoprotection is not a cure-all.=20
Putting the tissue in a -80C freezer is bad practice. Freeze very=20
rapidly with dry ice or 2-methylbutane cooled with liquid nitrogen, THEN 
put is a pre-cooled vial (or whatever) and put in the freezer.
    Also, immersion of a whole mouse brain will not provide good 
fixation of deeper structures. You are almost certainly getting poor=20
morphology that looks like freezing artifact. I suggest fixing by=20
perfusion or slicing the brain into 5 mm sections for fixation.

Geoff

Aine Behan wrote:
> I was looking for advice on troubleshooting already cryoprotected tissue for
> freezing artefact (FA). 
>
>  
>
> I have mice brains dissected out after cervical dislocation, postfixed in 4%
> Paraformaldehyde/PBS overnight (4 degrees), cryoprotected in 30% sucrose for
> ~24 hours (4 degrees) and then placed in -80 freezer. This FA never was an
> issue before and the animals are treatment free so anyone know why I would
> be getting this now?
>
>  
>
> Regards,
>
>  
>
> Áine
>
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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 
mcauliff@umdnj.edu
**********************************************




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