[Histonet] Re: Superfrost slides-Histonet question

[I.B.]

Sarah,

Our cryostat is a Leica 1850.  Cryosectioning has, unfortunately, been an
area of contention between myself and the person who trained me;
specifically the blade angle.  My trainer insists that the angle should
never been changed, mostly because it has produced good results for her.
However, the tissue I am sectioning is quite different and that angle
(negative two, oddly enough) produces sections that curl into a tight tube
and are therefore quite useless.  By playing with the angle I have found
that 3-4 degrees produces uncurled sections (more or less, they are often
still 'springy' ) that I can actually use and, for a while, everything
worked fine.  I realize this compresses the tissue more, but can it affect
adhesion as well?  I would like to think that my cryosectioning technique is
good, but I am of course rather biased and am always open to new and better
ways.  The Leica user manual is more of an Installation manual, and requests
for clarification from there website on several issues has gone unanswered.
Can you suggest a good source for cryosection technique and other
information?
Beyond that, I will try the paintbrush technique.  I always avoid touching
the tissue directly out of concern for damaging it, but at this point it can
not hurt.
There seems to be much praise for gelatin-subbing.  A fellow histonetter
sent me a protocol earlier, but if you can send me your favorite protocol
for comparison that would be great.

Ion

p.s.  Thanks to everyone who has answered so far.  Sorry I replied to
everyone backchannel, I did not realize that Gmail was sending my messages
to private addresses instead of the listserv.  Opps.


On 10/2/06, Pixley, Sarah (pixleysk) < PIXLEYSK <@t> ucmail.uc.edu > wrote:
>
>  Dear Ion:
>
> We have had similar luck with SuperFrost slides. We do brain and nose
> tissue, from rats perfused with 4% paraformaldehyde, 15-20% sucrose, frozen
> and cryosectioned. The tissues were not sticking to the slides during
> immunostaining. I have two suggestions, based on our experience: First, we
> did find that improving our sectioning significantly helped keep the
> sections on the slides. So you may want to check your cryostat and make sure
> it is cutting well (new blade or freshly sharpened, check knife angle, etc.
> and check out the sections right after fixing on the slide). If the sections
> do not stick down well immediately, or if they have air bubbles under them
> which cause slight wrinkles, then they can lift off in the immunostaining
> procedures. It turned out to be a combination of getting a new technician up
> to speed on sectioning (darn that learning curve!) and learning some new
> tricks for sectioning. Someone showed us that if you take a cold dry paint
> brush and lightly tap down the sections immediately after putting cold
> sections on a cold slide, then you can avoid air bubble problems and get the
> sections to stick better.  However, Second, we also just gave up on
> SuperFrost and went back to gelatin-subbing regular slides. That really
> helps, even if the sections are not cut well. If you need a subbing
> protocol, let me know.
>
> Sincerely,
>
> Sarah Pixley, Ph.D.
> Associate Professor
> Dept. of Cell Biology, Neurobiology and Anatomy
> University of Cincinnati College of Medicine
> Vontz Center for Molecular Studies, #3112
> PO Box 670521 (3125 Eden Ave)
> Cincinnati, OH 45267-0521
> Tel# (513)-558-6086
> Fax# (513)-558-4454
> sarah.pixley <@t> uc.edu
>