i need help for brain cryostat section

Henk Veldman H.Veldman at lab.azu.nl
Wed Mar 3 10:56:52 EST 1999


arnaud2507 at my-dejanews.com wrote:

> hi,
> I'm a little problem with brain cryostat section.
> I want to cut mice brain with a cryostat in order to stain with thionin and
> see the purkinje cells in the cerebellum (for count), but i don't know the
> better fixation procedure to cut the brain at 10µm max.
> For the moment, i froze the brain in isopentane. With this procedure, it's
> really difficult to section it under 25µm, and at this thickness, it's
> pratically impossible to counts the P.Cells, so my question is : is it
> possible to fixed the brain after frozen in isopentane in order to cut it at
> 10µm thickness?
> What is the good temperature in cryostat to cut brain tissu?
> If i fixed the brain in formalin, what is the procedure, after fixation, to
> cut it in cryostat?
> If someone have a good procedure, please help me!!!
> bye
> arnaud

There are two approaches to this:

1:
a - Fix the brain (preferably by vascular perfusion) with buffered
formalin (or other suitable fixative).
b - Infiltrate the brain with up to 25% sucrose (use several steps e.g.
7,5%, 15%, 25% in phosphate buffer, long enough to make the tissue
sink).
c - Freeze the brain (if possible using isopentane and liquid nitrogen)
d - Cut cryostat sections at -25°C.
e - Pick up sections on slides pre-coated with formol-gelatin (or other
suitable adhesive)
f - Let sections dry onto slides.

2:
a - Use fresh, unfixed brain
b - Freeze (as above)
c - Cut cryostat sections (as above)
d - Pick up on slides (need not be coated)
e - Let dry to attach
f - Fix sections in buffered formalin or other fixative

The first method is bound to give the better morphological preservation.
The second is somewhat more flexible (different postfixations on same
block, better enzyme and antigen preservation).
The concentration of the sucrose and the cutting temperature can (within
limits) be adapted to each other: the lower the temperature the higher
the sucrose concentration should be.
For some stains it will be advantagious to delipidate the sections (by
means of alcohol or acetone) before staining.

Succes

Henk



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