[Histonet] RE: cutting brain sections on the cryostat

From:"Pixley, Sarah (pixleysk)"

Dear Thomas Crowell:

I am not sure exactly what might be your problem, but I can tell you
what we do that is working, and may be slight changes compared to what
you are doing.

First, after perfusion with 4% paraformaldehyde in 0.1 M phosphate
buffer, we only postfix for 2-4 hours. Then we put the tissue (rat or
mouse brain) into 30% sucrose in 0.1 M phosphate buffer (PB) and leave
it for at least overnight (you don't say how long you leave it) or until
the tissue floats. I don't know if the PBS, with saline, vs. PB, or the
timing makes any difference. Then we use the Shandon (now Thermo) M1
embedding matrix instead of OCT. We have found the M1 to be preferable
to OCT in that there is less curling of the sections, better ease of
cutting. I would not leave the tissue in either matrix for very long; it
is not particularly good for the tissue. Your OCT compound may also have
gone bad. We have had the M1 matrix go bad after repeated use, in and
out of the refrigerator. When it goes bad, it does not freeze well and
remains soft and mushy around the frozen tissue. OCT may do something
else when bad.  

Your freezing method may also complicate matters. We have had trouble
using chilled isopentane with brains because the tissue cracks. Thus, I
speculate that it may possibly also cause contraction, away from the
OCT.  There were some recent good threads on this board about freezing
methods--I would recommend checking for those. We now freeze the tissue
in the cryostat by putting the tissue on the chuck and then adding dry
ice powder onto the tissue. Less cracking/distortion, and no other
problems so far. However, we haven't recently done whole brain that way,
only smaller slices.

Other than that, you might want to check your cryostat. The settings for
the knife angle, the temperature, the roll blade can all affect how the
sections come off and adhere to the slide.

Good luck!
Sarah Pixley
(p.s. thanks to all who are answering my question about fluorescent
cytoplasmic markers--I am going to check your suggestions out!)

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