[Histonet] Processing Question

[Herrick, James L. (Jim)]

Dear Histonet Colleagues,

I am trying to develop a processing protocol for our Leica TP1020, but
the first run has not been very successful. The specimen is canine femur
(section lengths of 1" to 1-1/2 "). Following is a list of the
processing reagents and times for my first attempt:
	
	70% ETOH for 4 hours with vacuum
	95% ETOH for 4 hours with vacuum
	95% ETOH for 4 hours with vacuum
	95% ETOH for 4 hours with vacuum
	100% ETOH for 8 hours with vacuum
	100% ETOH for 8 hours with vacuum
	100% ETOH for 8 hours with vacuum
	100% ETOH for 8 hours with vacuum	
	Methyl Methacrylate (Pure) for 4 hours with vacuum
	Methyl Methacrylate (Pure) for 6 hours with vacuum
	Methyl Methacrylate (Pure) for 8 hours with vacuum
	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
		(specimen was placed into refrigerator overnight)
	
	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
		(specimen was placed into refrigerator overnight)

	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
	Methyl Methacrylate with Dibutyl Phthalate and Perkadox for 4
hours with vacuum
		(specimen was placed into refrigerator overnight)

	Specimen was removed from refrigerator and embedded in a water
bath for 2 days.

I cut the specimen in half in the longitudinal direction. The cortices
appear to have been infiltrated fairly well, but the marrow still has
some very large holes. If anyone would happen to have a protocol that
works well with this type of tissue and this processor, it would be
greatly appreciated if you could offer your advice and expertise as to
what I should do next. I do have one more specimen still running in the
processor and will probably embed it on Monday. The first specimen was
started on a Monday and embedded the following Monday (approx. 8 days),
so this second specimen will have been in MMA for an extra 8 days before
embedding. Thank you very much for your help.

Jim