Re: sectioning of frozen tissues

From:"J. A. Kiernan"

Dale Osborne wrote:
> times the morphology of the sections (especially small tissues such as
> mouse thymus) are terrible.
>           1.Before cutting sections, allow the temperature of the block
> to equilibrate to the temperature of the cryostat (-20#176#C).

If this means freezing the tissue slowly, you will get
big ice crystal holes that completely distort the

>           2.Place the tissue block on the cryostat specimen disk. Adjust
> the positioning of the block to align the block with the knife blade.
> Cut tissue block until the desired tissue is exposed.
>           3.Cut sections of the desired thickness (usually 5 um), place
> the sections on a Fisher Superfrost slide and dry overnight at room
> temperature.
>           4.Fix slides by immersion in cold acetone (-20#176#C) for 2
> minutes or other suitable fixative (e.g. alcohol, formal alcohol,
> formalin, etc.), air dry at room temperature and proceed to staining
> (Section III).

Alcohol (and probably also acetone) at -20C will precipitate
proteins without denaturing them; that is, the soluble proteins
remain soluble and will redissolve in water when the section is
rehydrated. 0 to +4C is a more reasonable "cold acetone." Brief
immersion in a formaldehyde-containing liquid is pointless 
because the chemical reactions of formaldehyde with proteins
are quite slow. If you put unfixed sections into aqueous
reagents they well be damaged, with swelling of collagen,
extraction of cytoplasm etc. 

>           5.Alternatively, the frozen section slides can be stored for a
> short period of time at -70#176#C in a sealed slide box. When ready to
> stain, remove
>             slides from freezer and warm to -20#176#C in the cryostat or
> -20#176# freezer, fix for 2 minutes in cold fixative (acetone or other
> suitable fixative)
>             and allow to come to room temperature to continue with the
> staining.
>     Now, my question is wouldn't allowing the section to dry overnight
> at room temperature (as described in step 3) result in destruction of
> certain proteins (maybe ones we want to stain for)?

Simple drying might mildly denature some proteins or adversely
influence some enzymatic activities, but it won't alter their 
chemical reactivity. 

I think your problems are from slow freezing and non-fixation.

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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