[Histonet] Re: Superfrost slides-Histonet question

[I.B.]

Mari Ann,

The tissue is embronic Sea Lamprey, 5-7mm long and 1mm thick at best, cut at
-22oC in OCT compound (Tissue-Tec, Sukura Fintech Inc, I believe) to 10um.
I have tried 12 and 14um with similar results.

Ion


On 10/2/06, mari.ann.mailhiot <@t> leica-microsystems.com <
mari.ann.mailhiot <@t> leica-microsystems.com> wrote:
>
> Ion
>
> Can you tell me what type of tissue you are cutting and how thick you are
> cutting your specimen.
>
> Best Regards
>
>
> Mari Ann Mailhiot BA HT ASCP
> Application Specialist
> Leica Technical Assistance Center
> 800 248 0123 x7267
> 847 236 3063 fax
> mari.ann.mailhiot <@t> leica-microsystems.com
> www.leica-microsystems.com
>
>
>
>              I.B.
>              <beldorth.msu+his
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>                                        [Histonet] Re: Superfrost
>                                        slides-Histonet question
>              10/02/2006 02:03
>              PM
>
>
>
>
>
>
>
> 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
> >
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