[Histonet] Re: Superfrost slides-Histonet question

[I.B.]

Hi Gayle,

Looks like I have to eat my earlier words.  I was informed we use low
profile blades, though it turns out they are Leica 818 high profile blades.
Oh, if I could make a list of the things I was incorrectly taught . . .
Anyway, exactly how slowly do you turn the wheel?  You make it sound like a
three-toed sloth made it's way into a biology lab :0  This seems to be
another of those areas that depends on tissue and freezing media.  I used to
section adult Sea Lamprey brain & nasal epithelium surrounded by a good deal
of additional tissue, and the cutting stroke was very fast, not quite
violently fast, but quite fast.  My embryos get mad at me if I treat them
like that.  Has anyone ever considered converting this list to a Web Forum?
That way there can be some permanent posts stickied to the top dealing with
issues that tend to send newbies into convulsions.  I will play around with
things some more, but I am pretty sure I am as cold and slow as I can get.

Ion



On 10/2/06, Gayle Callis <gcallis <@t> montana.edu> wrote:
>
> Ion,
>
> I will go back and look at the blade holder again - angles will drive you
> nuts!!!
>
> When I finish the cutting stroke i.e. turning the flywheel, I generally
> stop near the top and NOT let the section release entirely from the block
> face.  I can maintain a flat section without curling this way, using the
> brush.
>
> I do not use low profile blades, they are too flimsy for my work, and high
> profiles are just as sharp, but sturdier, especially for thick or tougher
> tissue sections.  What blade are you using?
>
> We have use Tissue Tek Accuedge for years in our cryostats although I do
> NOT use them for paraffin.  I can't cut sucrose protected tissues at -22,
> just too warm - but it may be the nature of the tissue used too and mine
> is
> much different than your lamprey!  I crank it down to -25 or -26 for
> anything prefixed, sucrose protected or the sucrose oozes out like syrup.
>
> I section VERY slowly which helps keep that section attached at top of
> blade better.  In fact, I section slower than  most automated cryostats!
>
> Will let you know the angle numbers -
>
> Gayle (yeah, science is a hoot!)
>
>
>
> At 02:52 PM 10/2/2006, you wrote:
> >Thanks Gayle,
> >
> >I have actually been to Pathology Innovations and tried the brush
> technique,
> >and done correctly it does prevent tissue from rolling, at least as the
> >front edge comes off the block.  Often, however, the tissue will roll
> from
> >the back edge once it detaches.  Not always, but the nature of the tissue
> >(really thin) means that every section counts.  I may start trying it
> again,
> >however.  Also, you kinda lost me on the angles bit.  I counted 6 numbers
> >and none of them seem to add up :)  We use a low profile 'razor' blade
> and I
> >take the 0deg mark to be 0deg.  I set it to 3-4deg and 10um sections come
> >off flat (provided, of course, that the anti-roll plate is positioned
> >correctly).  Well, actually sometimes a bit wavy, but at least not curled
> >into a tube.  More or less angle and they curl.  It sounds like you get
> good
> >results with a greater angle, and others have called for less.  This
> seems
> >to be one of those indeterminate situations that depends more on what
> works
> >in a given setting for a specific tissue than what is correct or
> incorrect.
> >Gotta love science . . .
> >
> >Ion
> >
> >
> >
> >
> >On 10/2/06, Gayle Callis <gcallis <@t> montana.edu> wrote:
> >>
> >>Ion,
> >>
> >>We have three 1850's and it is possible blade angles can be a bit
> >>different
> >>for thicker sections, the different types of blades one uses (high
> versus
> >>low profile) and also differences between different blade
> >>manufacturers.  Compression generally is NOT good, as it is hard to get
> >>the
> >>section flat once it is compressed and then have it be flat on a
> >>slide.  When doing the brush technic, use a sable brush #1 or #2, and
> make
> >>sure the tissue is surrounded by some OCT so you can grasp the frozen
> OCT
> >>gently instead of the tissue.  It is possible to use brush on unembedded
> >>tissues but you can't be a Picasso (actually brushing/touching the
> tissue)
> >>but rather let a few bristles guide the section onto the knife
> holder.  Go
> >>to Pathology Innovations website and watch Dr. Peters use the brush
> >>technic, he does it as well as anyone around.   I haven't used an
> antiroll
> >>device for years with exception of 50 um prefixed tongue - sometimes I
> >>have
> >>to use it.  We set our blade angle just one mark or 6 towards the 10
> mark,
> >>5 being the middle.  This would be an 11  to 12 degree angle.  5 to us
> >>means 10 degrees, and I must admit, angles are sometimes hard to
> >>understand
> >>from one cryostat to the next.
> >>
> >>Your Leica representatives, often locally or even experts in their main
> >>office, can be of help too.
> >>   mari.ann.mailhiot <@t> leica-microsystems.com or
> >>jan.minshew <@t> leica-microsystems.com are two ladies who will steer you in
> >>the
> >>right direction, both are experts at microtomy/cryomicrotomy and sell
> >>Leica
> >>1850's.
> >>
> >>Good luck on your dilemma
> >>
> >>Gayle Callis
> >>MT,HT,HTL(ASCP)
> >>Research Histopathology Supervisor
> >>Veterinary Molecular Biology
> >>Montana State University - Bozeman
> >>PO Box 173610
> >>Bozeman MT 59717-3610
> >>406 994-6367
> >>406 994-4303 (FAX)
> >>
> >>
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>