[Histonet] HELP, Plastic sectioning

[Jorge Tornero]

2006/2/14, Jorge Tornero <jorge.tornero <@t> gmail.com>:
> Hi,
>
> I think the first thing you have to change is your knife. As far as I
> know, it must be a tungste-carbide knife (or glass).
>
> I use a leica rm-2145 microtome whit a D-profile knife. I cut with an
> angle of about 6 degrees.
>
> But also I have suffered from shattering and I was annoyed because we
> even use a leica EM-TP tissue proccessor to do the embedding. We use a
> very long protocol for dehydration and embedding (24 EtOH 70, 24 h
> EtOH 96, 24 h EtOH 100, 48 h 1:1 and 60 hours of pure activated resin)
> and it worked for everybody except us. Why? One day I realized that we
> put the pure resin and the 1:1 solution at the begginning of the
> proccess (when we started the EM-TP) and oviously the resin was
> damaged (it turns with a brownish color) when its turn in the
> processor began. So now, we put the resin into the processor hours
> before its use (because it is friday and we dont work on weekend and
> the dwarf inside the processor works for us and changes the liquids)
> And now everything is fine. Check if there is a failure in your
> protocol or equipment and remember to store any resin or its solutions
> in the fridge at 4%. Our problem was caused because the EM-TP only
> refrigerates the vial that is being used in that moment. So we trust
> that we had 4 ºC in our processor but it only was in the vial that was
> running.
>
> Saludos,
>
> Jorge Tornero
> Instituto Español de Oceanografia
> Cádiz - SPAIN
>
>
> 2006/2/14, Pamela Marcum <pmarcum <@t> vet.upenn.edu>:
> > At 01:57 PM 2/13/2006, Sunhwa Lee wrote:
> > >I am trying to do plastic sectioning with Fish brain and the section
> > >is shattered a lot.  I've been changing thickness 1-5um, new knife,
> > >and embedding materials (JB-4 and Technovit 7100), but not thing
> > >works. My slicing conditions are as follows.  Any suggestions, please.
> > >  In advance, thank you so much.
> > >
> > >1. Knife : High profile stainless steel knife
> > >2. Microtome: normal rotating Microtome (Leica 820-II)
> > >3. Thickness: 1-5um
> > >4. Cutting angle: 2-3degree
> > >5. Embedding material: GMA (Technovit 7100)
> > >6. Sample: fish brain
> > >
> > >_______________________________________________
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> >
> > Hi Sunwha,
> >
> > You did not mention how you are fixing and dehydrating the tissues and that
> > can affect sectioning also.  What is you schedule and how are you
> > polymerizing?  Is it room temperature or 4C?  You thickness with the
> > correct knife and tissue preparation is not the problem.
> >
> > As you are using JB4 or Technovit 7100 it will still require a glass knife
> > or tungsten carbide knife at the least for your best sections.  That may
> > require a special knife holder for the older blade types as in
> > pre-disposable.  I know Linda Jenkins has used disposable knives however, I
> > believe they were the low profiles.  Plastics are simply too hard for most
> > disposable knives and the knives will deteriorate too quickly for you get
> > good sections routinely.  A standard microtome is usable just not
> > recommended as they are not generally heavy enough to cut smoothly with
> > very hard materials.  I have seen people with tons of experience section
> > well on lighter microtomes and I have done some it is just much harder to
> > get consistent sections.  Angle is determined by trying different positions
> > and seeing what works best.  One angle is not good for all blocks.  I
> > change angle sometimes when the block is larger or smaller and it is always
> > while I face the block so I don't lose sections of my tissue.  Hope this helps.
> >
> > Best Regards,
> >
> > Pamela A Marcum
> > Manager, Histology Special Procedures
> > University of Pennsylvania
> > School of Veterinary Medicine
> > R.S. Reynolds Jr.  CORL
> > New Bolton Center
> > 382 West Street Road
> > Kennett Square, PA 19348
> >
> > Phone - 610-925-6278
> > Fax     - 610-925-8120
> > E-mail - pmarcum <@t> vet.upenn.edu
> >
> >
> >
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>