[Histonet] freezing artifact

[Geoff McAuliffe]

    You are freezing too slowly. Cryoprotection is not a cure-all. 
Putting the tissue in a -80C freezer is bad practice. Freeze very 
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 
morphology that looks like freezing artifact. I suggest fixing by 
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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>   


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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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mcauliff <@t> umdnj.edu
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