[Histonet] uneven alternating sections on cryostat

Thu Aug 27 18:21:14 CDT 2009

   Thanks,  Joe  and  Adam. I'm always paranoid about over tightening and
   warping  some  piece of the equipment but maybe I'm being a little too
   gentle.  I'll  make  sure  everything  is nice and snug and see how it
   goes.
   Happy slicing...
   Nate
   Galbraith, Joe wrote:

Nathan:

As indicated by others - first check that everything is tight as that is
most often the issue - don't forget to check the adhesion of the
specimen to the chuck as well.  Also check the angle of approach to the
blade as it could be too steep or too shallow.

If everything is tight and properly adjusted, then you may have a
retraction issue.  If your cryostat retracts during the upstroke, the
head may still be returning to the proper distance from the head to the
specimen during the downstroke and hence you may see no section at first
and then a gradually increasing thickness of section during the
remainder of the downstroke.  This can be due to two causes: 1) You can
make this happen by operating the microtome at too high of a speed, if
you slow down and the process improves then speed may be a factor; 2)
Your microtome may be sticky and require maintenance (defrosting,
cleaning, lubrication, etc.).  The latter is best left to someone with
extensive experience or a service rep.

Good luck.

Joe Galbraith
University of Iowa
[1]joseph-galbraith <@t> uiowa.edu

-----Original Message-----
From: [2]histonet-bounces <@t> lists.utsouthwestern.edu
[[3]mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Adam .
Sent: Thursday, August 27, 2009 4:53 PM
To: Nathan Cramer
Cc: [4]histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] uneven alternating sections on cryostat

I was having this problem too, but I think I finally figured it out.
There
is a screw on our cryostat that attaches the chuck to the rest of the
machine. This screws fits into a small hole in the chuck. Sometimes the
screw isn't well set in the hole or is well set but for some reason
comes
loose and causes the chuck to wobble. For some reason, this causes your
problem. Now I make sure that everything is tightly secured, and I
rarely
have this problem.

Good luck,
Adam

On Thu, Aug 27, 2009 at 4:47 PM, Nathan Cramer [5]<natecrmr <@t> gmail.com>
wrote:

  Thanks, Robyn... I'll make sure the holder is clamped down.

(sometimes

  its the little things...)
  Best
  Nate
   R J VAZQUEZ wrote:

    Nathan,
    It sounds like the blade holder is not secure enough or even in

the

    tightness on each side.
    Hope this helps.
    Robyn

    > Date: Thu, 27 Aug 2009 16:44:11 -0400
     > From: [[6]1]natecrmr <@t> gmail.com
    > To: [[7]2]histonet <@t> lists.utsouthwestern.edu
     > Subject: [Histonet] uneven alternating sections on cryostat
    >
    > When cutting PFA fixed, cryoprotected tissue on our cryostat, I
    >  frequently  find  that  every other section gets cut

improperly.

    I'll get
    >  one nicely cut section and then on the next pass I only get

half

    of a
    >  section. This cycle simply repeats over and over and I lose

many

    slices.
    > It has been a while since our cryostat has been serviced, so I'm
    >  wondering  if  this  an  operator  error or a machine problem.

I

    thought
    >  maybe  the  tissue  temperature hadn't settled properly, but

the

    uneven
    >  cutting  still  happens  even  when  I let the tissue sit for

30

    minutes in
    > the chuck holder. (cutting mouse spinal cord at -20C)
    >
    >  On another note, if anyone has any tips for improving

white/gray

    matter
    >  contrast  in frozen spinal cord sections stained with luxol

fast

    blue,
    >  I'd  be very appreciative. I do defat the slices with

chloroform

    and
    >  differentiate  with  lithium carbonate but everything is

usually

    either
    >  very  dark  or  very  light. I am learning as I go (checking

the

    archives
    > here often) so any help would be great.
    >
    > Thanks!
    >
    > Nathan Cramer
    > Neurobiology and Behavior
    > Cornell University
    > Ithaca, NY 14853
    >
    > _______________________________________________
    > Histonet mailing list
     > [[8]3]Histonet <@t> lists.utsouthwestern.edu
    > [4][9]http://lists.utsouthwestern.edu/mailman/listinfo/histonet

References

  1. [10]mailto:natecrmr <@t> gmail.com
  2. [11]mailto:histonet <@t> lists.utsouthwestern.edu
  3. [12]mailto:Histonet <@t> lists.utsouthwestern.edu
  4. [13]http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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References

   1. mailto:joseph-galbraith <@t> uiowa.edu
   2. mailto:histonet-bounces <@t> lists.utsouthwestern.edu
   3. mailto:histonet-bounces <@t> lists.utsouthwestern.edu
   4. mailto:histonet <@t> lists.utsouthwestern.edu
   5. mailto:natecrmr <@t> gmail.com
   6. mailto:1]natecrmr <@t> gmail.com
   7. mailto:2]histonet <@t> lists.utsouthwestern.edu
   8. mailto:3]Histonet <@t> lists.utsouthwestern.edu
   9. http://lists.utsouthwestern.edu/mailman/listinfo/histonet
  10. mailto:natecrmr <@t> gmail.com
  11. mailto:histonet <@t> lists.utsouthwestern.edu
  12. mailto:Histonet <@t> lists.utsouthwestern.edu
  13. http://lists.utsouthwestern.edu/mailman/listinfo/histonet
  14. mailto:Histonet <@t> lists.utsouthwestern.edu
  15. http://lists.utsouthwestern.edu/mailman/listinfo/histonet
  16. mailto:Histonet <@t> lists.utsouthwestern.edu
  17. http://lists.utsouthwestern.edu/mailman/listinfo/histonet