[Histonet] Over-processing of brain tissue

[Rene J Buesa]

It seems to me you are processing too much unless the slices are 3mm thick or more.I suggest you to cut the dehydration to 45 minutes (the sequence seems OK)Reduce the pure 2-propanol to just 2 changes (30 min is OK)Add 1 change of a mixture 1:1 of 2-propanol and xylene + 2 xylene stepsthen to paraffinUse vacuum and mixing/agitation in all steps (it will help)If you want really to simplify, use 2-propanol in all steps instead of ethanol and eliminate the xylene. You can go from pure 2-propanol → 1:1 mixture of 2-propanol with paraffin → 3 paraffin changes.You will eliminate xylene and obtain very good results.Contact me if you need some publications on this procedure.René 

    On Tuesday, June 14, 2016 9:53 AM, "Wheelock, Timothy R. via Histonet" <histonet at lists.utsouthwestern.edu> wrote:
 

 Good morning everyone:

I seem to be having problems with over-processing of brain tissue.
I use a VIP6 processor.
I start off with 1 hour each of 30%, 50%, 80%, and 95% isopropanol.
Then half-hour each of three changes of absolute isopropanol.
Then half hour each of three changes of xylene.
Then half-hour each of 4 changes of Paraplast.

I use a slow mixing cycle and no vacuum-pressure for all reagents except the paraffin.
For the paraffins, I use vacuum pressure, but no mixing cycle.

I rotate the paraffins during each run of tissue.
I replace the dilutions of isopropanol after I have processed 500 blocks.
I rotate the absolute isopropanols and xylenes after 500 blocks as well.

When I embed the brain tissue, it sometimes seems a little stiff, or even a bit brittle, to one extent or another.
When I trim the blocks, they seem somewhat dry. Once in a while there is even a saw-dust effect.
Before I section the blocks, I have to keep them on ice for at least 3 hours before they are moist enough to cut.
Even then, the first case (out of 5 cases) shows chatter in the cerebral cortex.
When I examine the stained sections microscopically, even the best sections look a little "rough" or dry.
Taking microscopic images can be difficult at 40x ( or even 20x) because all this roughness shows.
The brain tissue looks "granular" rather than smooth, especially with a LHE stain.

I have continued to reduce the times to their present values, but it still does not seem enough.
I absolutely love the VIP6, but it is a much more powerful machine than the Shandon Hypercenter XP that I use to have.
Perhaps I have still not fully compensated for this power.
It has 4 paraffin reservoirs rather than 2 on the old Hypercenter, and the reagent reservoirs hold twice as much volume.

Any ideas on how to resolve this problem?
Should I reduce the times further?
Should I alter the use of the mixing and/or vacuum-pressure?

Thanks for any help that you can give me.

Tim


Tim Wheelock
Assistant Director, Neuropathology
Instructor of Neuroanatomy
Tour Coordinator
Harvard Brain Tissue Resource Center
Room 203, Mailman Research Center
McLean Hospital, Belmont, MA 02478
Phone: 617-855-3592
Cell:    857-234-9311
Fax:    617-855-3199



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