[Histonet] Summary of hand-processing/vacuum advice given to the Rookie

From:"Jack England"

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* 
correct me):

--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 
melting point.

--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,
Jack England

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