Re: [Histonet] transferring paraffin ribbon

From:"Jacqueline Miller"

Hi everyone,

I just wanted to say thanks to everybody who gave me suggestions/advice
for transferring paraffin ribbons to the water bath.  Some hints that
proved particularly helpful were 1)  Keeping the blocks cold--virtually
eliminates wrinkles 2) Simply making sure that the plate of the knife
holder and the knife itself are very, very clean.  This helps in getting
smooth, unwrinkled sections off of the knife  3) Keeping the pair of
forceps used to remove the sections ice cold and keeping them very, very
clean.  This helps keep the paraffin from sticking to the forceps, thus
allowing that first section to be kept if necessary.

My sectioning is going much, much better, so thank you all very much.

Now, if someone could solve my chatter problem on the cryostat.....
It has all the service techs, including a specialist who flew in from
California to look at it today, baffled.  They're saying it might be the
bearings, but they just don't know.  Basically, the chatter occurs when
cutting at a fast speed, and hardly at all when cutting slowly.  We've
tightened just about every nut, bolt, and lever on the cryostat, tried
different blades, adjusted the clearance angle, and we're running out of
ideas.  It's a Leica CM1850.  Any ideas are greatly appreciated.


>>> "Sarah Jones"  08/01/04 2:11 PM >>>
Hi Jackie,
   I use a wooden applicator stick that I angle cut, at the tip, with a
single edge razor blade, making a fine point.   I dip the tip of the
stick into my waterbath before touching the ribbon and the water makes
the ribbon adhere to the stick.  I use them for both the beginning and
the end of the ribbon.  When doing serial sections, it's difficult to
save every section if you need more than one ribbon.  At the best of
times, I'll usually lose one section at the beginning and one at the
end.  If I have trouble getting the ribbon started, I may lose even more
than that.  If researchers are doing 3D reconstruction, I just keep a
record of where sections were lost and how many sections were lost.  I'm
doing that right now with some laser induced retinal lesions in the eye
of the rat.  
  If you need thin sections (3-5 microns), the blocks need to be cold. 
If the block isn't cold, the ribbon will be compressed.  I always cut
thin sections off of an ice tray.  I use the dissecting pans (without
wax) available from Fisher for my ice trays.  If you are doing thicker
sections, you can cut at room temperature.  
  I once had a disposable knife holder where the sections would stick
to the metal.  I ended up putting a piece of wide, clear, plastic
packaging tape over it and that kept the ribbon from sticking to it. 
You might try Paraguard too, a spray used for keeping paraffin from
sticking to surfaces.  It has a nasty smell that makes me nauseous so I
don't use it myself.  Also, make sure there isn't any water on the face
of the block or on the knife before you start a ribbon.  

Sarah Jones HT(ASCP)
Dept. of Vet. Anatomy & Public Health
Histology Lab
Texas A&M University
College Station, TX 77843-4458
phone: 979-845-3177
fax:  979-458-3499

>>> "Jacqueline Miller" 
7/30/2004 2:16:00 PM >>>
Hi everyone,

I would like to know about the techniques that histotechs use to cut
paraffin sections--especially how the ribbon is transferred from
microtome to water bath while preserving the first and last sections.

I've been sectioning paraffin blocks for almost 4 years now.  However,
I've never needed to  cut serial sections and the samples were always
large.  So, I never worried about losing the first section of each
ribbon (and sometimes the last).  I would like to know what techniques
the histologists out there use to transfer the ribbon from the
to the water bath.  I've used metal forceps and my fingers (gloved and
not gloved) to grab the first section (but the section sticks to the
forceps), and I'm using the wooden part of a cotton swab to lift off
last section, which is working fairly well.

I'm also using a new microtome, Leica RM2235, and having some trouble
with the first section coming off the knife just wrinkling up.   And,
the surface below the knife is such that even if I grab the edge of
section with a paintbrush, it won't budge.  Is there anything I can do
to correct these problems?  Also, which is preferred, to cut the
chilled or to leave them RT.  I used to cut blocks chilled all the
but here it's preferred that I cut them RT unless I have a problem.  


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