Re: [Histonet] LINES IN PARAFFIN SECTIONS

From:Jan.Minshew@leica-microsystems.com

Hi Tim,

You have gotten some excellent suggestions so far, but I thought I might
ask a few more questions to see if the problem can be isolated a little
better.  Like Barry, I don't think the problem is caused entirely by the
disposable blades or the paraffin.  Unfortunately, there are a LOT of other
things to consider.

Do you turn your paraffin reservoir on the embedding station off and on
daily?  If so, it can cause problems if the paraffin does not have time to
melt completely because different additives have different melting points
and come out of the solid block at different times.  If you want to save
energy and turn it off when you are not using it, use as little paraffin as
possible so it will melt completely in the allotted time.  As Gayle
suggested, you should always stir the paraffin right before you use it to
evenly distribute all of the constituents.

Do you embed using cassettes?  I know this sounds odd, but if your
cassettes are not filled up high enough with paraffin the front portion of
the block (the part with the specimen) can become loose and have just
enough movement to cause problems.  If you are making blocks (not using
cassettes) are you clamping down directly on the paraffin.  Tightening down
directly on the paraffin can sometimes cause sectioning variances,
especially if you tighten really hard.  It's better to mount the block on a
wooden, plastic or metal plate and clamp down on that.

Are you fixing in 10% neutral buffered formalin?  If so, are you going from
fixative into an alcohol that is 70% or less in strength.  Higher
concentrations can cause the buffer salts to precipitate in your solutions
and the tissue.  This can make your blocks look like shredded wheat when
you section them.

Which microtome do you use...how old is it...and how well is it maintained?
Have you thoroughly inspected the front and back pressure plates on the
blade holder?  If there are any irregularities or uneven wear, it will
cause uneven pressure on the blade and produce uneven sectioning like you
described.  Does the pressure plate have an adjustment screw (or two) that
could be improperly aligned?

How much pressure is being exerted on the blade?  People think that if a
little is good then a lot is better, but that's not correct in this
instance.  The pressure plate should rest evenly across the blade (the
blade should be centered under the plate if possible) and only a slight
amount of pressure should be applied--just enough to GENTLY hold the blade
in place and not enough to cause the blade to bow.  Too much pressure will
also cause the problems you described.

Are all of the locking mechanisms on the microtome, knife holder base,
disposable blade holder, x-y orientation device and specimen holder locking
correctly?  Do the locking levers feel like they are locking properly--not
rotating past the locking point if you push them and not stopping in their
rotation before you feel they should?  Can you physically move any of the
pieces when they are locked down?

Please feel free to contact me or one of Leica's excellent Technical
Applications Center team members at 800.248.0123 if you are unable to
resolve your problem or have any further questions.  There are a lot of
things that can go wrong and cause sectioning problems and we would be
happy to help in any way we can.

Best wishes,

Jan Minshew, HT(ASCP)HTL
Marketing Manager
Leica Microsystems, Inc.
2345 Waukegan Road
Bannockburn, IL 60015
800.248.0123 x7051 toll free
847.405.7051 direct
847.405.6560 fax
jan.minshew@leica-microsystems.com




                                                                           
             Tim Wheelock                                                  
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                                       [Histonet] LINES IN PARAFFIN        
                                       SECTIONS 2                          
             01/17/2007 03:57                                              
             PM                                                            
                                                                           
                                                                           
                                                                           
                                                                           




Hi Barry:

I am cutting blocks that can go up to twice as large as a postage stamp,
although some are half that size or less.
The brain tissue is somewhat heterogeneous in density because of the
variable distribution of gray and matter in the same block.

I am also going to try blank blocks, first thing in the morning,  to see
if there is something in the paraffin.

Tim


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             Tim Wheelock                                                  
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             Sent by:                  Histonet@lists.utsouthwestern.edu   
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             ern.edu                                               Subject 
                                       [Histonet] LINES IN PARAFFIN        
                                       SECTIONS                            
             01/17/2007 03:02                                              
             PM                                                            
                                                                           
                                                                           
                                                                           
                                                                           




Hi Everyone:

I have a serious problem with lines/splits forming in my 5 micron
sections as they come off the microtome.
I just started using Paraplast Xtra Tissue Embedding Medium.
My infiltrating wax is regular Paraplast.
My disposable blades are Surgipath high profile Teflon coated blades.
The microtome is a Microm HM 355S.
I cut human brain tissue exclusively.

I had been using Surgipath embedding media.
I decided to try the X-tra because I had been having problems with the
sections being thick and thin on just one side of the section.
Also the wax seeming to be thicker (more opaque) on one side of the block.
The blocks of tissue also were not laying flat in the mold, hence a lot
of trimming after.
There was some problem with lines even with the Surgipath but it was
intermittent., and not nearly as bad as the X-tra.

I notice that trimming requires more physical effort with the Paraplast
X-tra.
And I have had much more of a problem with lines than with the Surgipath
Embedding Media.
Does it have a higher % of plastic in it.

I cleaned out the reservoir on the embedding center and played with the
knife angle but to no avail.
I am not sure if the lines have to do with the wax or the blades or both.

Thank you for any advice you can give me.

Tim Wheelock
Harvard Brain Tissue Resource Center
McLean Hospital

I





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retransmission, dissemination or other use of or taking of any action in
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