Re: bovine tendon processing - some ideas

From:"J. A. Kiernan"

> "Bellantoni, Rose" wrote:
> processing cross sections of tendon at 20 minutes each in 7
> stations of graded alcohols, 20 min. each in 2 xylene stations,
> and 35 min. each in 4 paraffin stations.  The tissue is fixed
> overnight in 10% NBF. ...
>  Before sectioning the blocks are soaked in Mollifex ...
> ... remains as hard as a rock ...

I've never worked with bovine tendons, so probably
should not be offering suggestions. For other hard
objects, however, I've found that sectioning is easier
after a clearing agent that is more viscous or "oily"
than xylene. Of the ones I've tried, terpineol and
cedarwood oil have been better than methyl salicylate,
methyl benozoate or benzyl benzoate. They need plenty
of time to penetrate (such as 2 changes each 12 hours
for an object 5 mm thick), and lots of time infiltrating
with wax (such as 4 changes of 1 or 2 hours each). 
Some of the oily clearing agent remains in the specimen
even with these long times in melted wax; you can smell
it on the cut surface of the block. I suspect that it
may provide some internal lubrication for the passage
of the knife through tough tissue. (Is this hypothesis

The times etc in the preceding paragraph are for manual
processing, with the volume of solvent or wax being
about 20 times the volume of the specimen. With cedarwood
oil (expensive) I re-use the 2nd change once, as a 1st
change, collecting it in a "once used" bottle. I do the
same with terpineol (less expensive and probably just as
good, but tradition has it that cedarwood oil should be 
used if you want to see things like mitochondria and
some kinds of secretory granules in paraffin sections;
I'm not convinced it's any different.) Terpineol and
cedarwood oil have quite pleasant smells. Terpineol is 
"synthetic oil of lilac," but it doesn't smell like 
lilac (Syringa spp) flowers. Among the oily clearing 
agents listed in the previous paragraph the only 
nasty-smelling one (to my nose and rhinencephalon) is 
methyl benzoate. 
If you have to worry about disposing of these viscous
clearing agents, there shouldn't be a serious problem.
None are considered seriously toxic. They are all
flammable if sufficiently heated, yielding water and
carbon dioxide, and can be put in a a waste drum for
non-chlorinated solvents, destined for incineration.

All the foregoing remarks relate to a possible easy
solution to your problem. You will certainly get advice 
from others who will advise embedding in plastic, and
that may well be what you will have to do, especially
if thin sections are needed. Nitrocellulose and
nitrocellulose-paraffin double embedding are other
things you could try.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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