[Histonet] Summary of hand-processing/vacuum advice given to the Rookie
Thanks so much to all for your advice; we used the infiltrator yesterday and
are eager to see how everything pans out.
In case someone else out there is facing a similar situation, here's the
advice that came back (for those zen masters among you, if any of my
information here is incorrect or needs clarification, by all means *please*
--Consult a good textbook. Recommended in particular was Kiernan, J. (1999),
Histological & Histochemical Methods, 3rd Ed, Arnold, ISBN: 0750649364.
--Samples should be cleared for no more than 2 hours in room temp. xylene
prior to infiltration, with the xylene changed out at least once, and all
solution containers having volumes well in excess of the tissue being
--After coming out of xylene, go through at least one change of paraffin
prior to infiltration (we're use three baths, then proceeding to the vacuum
bath). Make sure that paraffin baths are of a fairly large volume relative
to the tissue being infiltrated, so that the vast majority of xylene can be
displaced with paraffin prior to heated vacuum. Also make sure that the
paraffin used for infiltration is no greater than +2 degrees C of the
--For the truly ambitious, vacuum can be applied to each of the multiple
paraffin baths, provided that either the wax is changed between baths or
there are multiple baths used.
--Amount of vacuum to be applied is more art than science (as I'm learning
are so many things about histology...*grin*). 40-200mm Hg seems to be
acceptable, but trial and error seems to be the best way to find the sweet
spot. Likewise, the amount of time under vacuum seems to be highly variable;
we're using 1 hour under vacuum for our samples, but up to 4 hours was
recommended as well. Again, trial and error is the way to go.
--After infiltration, release vacuum SLOWLY so as not to a) gum up the
needle valve in the infiltrator, or worse b) spray paraffin all over the
There was a lot more advice, and I can't tell you all how much I greatly
appreciate you offering it. I was expecting a lot harsher response than I
got ("take a class, buddy!" comes to mind), and I'm amazed by the support
you've all provided. Hopefully down the line I'll be able to help out
someone else the same way you've helped me.
--Best Regards to all,
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