Re: [Histonet] Re: Superfrost slides-Histonet question
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.
On 10/2/06, firstname.lastname@example.org <
> 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
> email@example.com> To
> Sent by: firstname.lastname@example.org
> histonet-bounces@ cc
> ern.edu Subject
> [Histonet] Re: Superfrost
> slides-Histonet question
> 10/02/2006 02:03
> 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
> good, but I am of course rather biased and am always open to new and
> ways. The Leica user manual is more of an Installation manual, and
> for clarification from there website on several issues has gone
> Can you suggest a good source for cryosection technique and other
> 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
> 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.
> 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@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,
> > and cryosectioned. The tissues were not sticking to the slides during
> > immunostaining. I have two suggestions, based on our experience: First,
> > did find that improving our sectioning significantly helped keep the
> > sections on the slides. So you may want to check your cryostat and make
> > it is cutting well (new blade or freshly sharpened, check knife angle,
> > and check out the sections right after fixing on the slide). If the
> > do not stick down well immediately, or if they have air bubbles under
> > which cause slight wrinkles, then they can lift off in the
> > procedures. It turned out to be a combination of getting a new
> > 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
> > 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
> > 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
> > email@example.com
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