[Histonet] PARAFFIN SECTIONING ADVICE SUMMARY

[Tim Wheelock]

Hi everyone:

Thank you for the advice you gave me concerning the sectioning problems 
that I have been having recently.
I am beginning to systematically use them.

In the meantime, I thought that I would summarize them by quoting them 
in roughly the order that I received them.
Hopefully, this synopsis will help other people as well.

Thanks again,

Tim Wheelock
Harvard Brain Bank
McLean Hospital
Belmont, MA
617-855-3592



> *PROBLEM*
>
> 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.
>
>
> *REPLIES*
>
>
> 1. I believe that it may be a blade problem. You may want to try 
> switching blades.  Currently I am using the Extremus blade from 
> Sturkey.  It has worked wonderfully for me.  I also use the Paraplast 
> extra wax for embedding (Plus for processing).  How do your brains 
> looks - do they cut well other than the lines in the wax??  Just to be 
> sure it isn't a processing problem.
>
>
> 2. I would try first changing to another brand of disposable (JUST to 
> see if there is a difference).
> If the problem persists I would return to your usual disposables and 
> then try adding some bees wax (5%) to your paraffin (JUST to see if 
> there is a difference).
> If the problem remains it could be some dust in the paraffin or in the 
> molds; have you had problems with the air filters in your lab?I also 
> cut human brain tissue exclusively. For the past 15 years, I have been 
> using
>
> 3. TissuePrep 2 from Fisher. It cuts well and is problem free. You 
> could check and see if they would send you a sample.
> I use DuraEdge blades on an old Leitz microtome.
>
> 4. I started a new job 6 month ago, and we use the same parrafin and 
> microtome here as you do.   I find the parrafin to be so much better 
> than the Surgipath. We routinely cut at 3 microns with no problems at 
> all. This parrafin seems to be a bit more "sticky", but it's worth 
> it.  I wonder if the problem doesn't come from the microtome.  Did you 
> try cutting an empty block? We do this to check / reset the angle on 
> our microtome, and it's also useful to see if you have "grit" in the 
> parrafin.
    5. One sure-fire way to determine if the lines are the result of 
your disposable blade is to change blocks and see if the lines are in 
the same place on the block/within the ribbon.  Try a few blocks.  If the
         lines are in the same place, most likely the problem lies with 
the blade.  If the lines show up elsewhere, then it's probably not the 
blades (it's probably something in the paraffin).You can make up a few
         "blank blocks" (i.e. no tissue) to make it a little easier to 
see if the lines stay in the same position or if they change.  Sometimes 
tissue can contain natural components that will cause splits in your ribbon
         (for example, areas of microcalcification).  Using a blank 
block will eliminate that possibility.
>
> 6. Stir the paraffin just before embedding to redistribute the plastic 
> polymers and other additives in paraffin.  These tend to settle out at 
> melting, being heavier - a cheap spatula does the trick.
>
> 7. Forgot to mention - since you use two different types of paraffin 
> (one for infiltration, one for embedding), try to observe if the lines 
> or splits occur where the actual tissue is or whether they occur 
> around the periphery.  If, when you change blocks, you observe that 
> the lines move...and you consequently determine that it's not your 
> blade...look to see if the lines/splits are predominantly in areas 
> around the tissue or in areas within the tissue itself.  That may help 
> narrow down whether it's the embedding paraffin or the infiltration 
> paraffin, respectively.
> If you deduce it to be the paraffin, I would contact the 
> manufacturer.  Your cutting angle should have nothing to do with the 
> artifact.  It's usually a manufacturing issue with the blade, a 
> manufacturing issue with the paraffin, debris in the wax baths of the 
> processor or the holding tank of the embedding center, or something 
> inherent in the tissue itself (I'm not an expert at cutting brain 
> tissue because I only did so every once in a while, so I'm not sure if 
> the issue would be something within the tissue itself).
>
> 8. 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?
>
>
>
> 9. Sometimes it is the temperature of the block when using paraplast.  
> If the block is too cold it will give thick and thin sections.  Try 
> warming the block up a little and see if that helps.  I just set a  
> couple of blocks on top of the microtome by the time they are cut they 
> are warmer.  Trim the block and then rub your finger over it to warm 
> it up.
> These things have worked for me, I use Paraplast for embedding.
>
>
> 10. Sounds to me that you have a cutting problem or problems, with 
> thickness variation and lines.
> I am assuming that your tissue is relatively uniform in consistency?
>
> I suggest problem might be one of the following.
> 1.    Is the knife securely clamped into the holder? If there is any 
> movement at all on one side this might explain the thickness differences.
> 2.    Does the holder have wear on one side, either the knife holder 
> or the sliding mechanism it sits on? 3.    Are these blades reliable 
> as regards having a sharp edge along entire edge? Rene's suggestion 
> should take care of this.
> 4.    I do not believe that the wax is the problem unless you have 
> poorly mixed wax or separation of the components. If this is the 
> problem then this effect should be seen on different sides of 
> different blocks in a random manner.
> If this wax is too hard then Rene's suggestion of beeswax should help
> Would you be kind enough to let us know what size blocks you are cutting?
>
>
>
>
>



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