Tim Wheelock twheelock <@t> mclean.harvard.edu
Fri Jan 12 12:54:41 CST 2007

Hi Thomas:

I stain 5 micron paraffin sections of human brain with Luxol Fast Blue 
Forget the Lithium.
The best differentiator is a 1% hydroquinone, 5% sodium sulfite aqueous 
This will pull out the Luxol from everything except the white matter, 
lipofuscin, and elastic fibers in arteries.
Works every time.

At least with my thin 5 micron sections, you only need 2 dips in the 
Then immediately into 3 changes of  tap water of about 20 dips each, 
once more in tap water for 1 minute, and then a final holding dish with 
tap water.
Your 7 micron sections should behave the same. If you want to add 1 more 
dip in the differentiator that should be fine too.

As for the pink stain that you have seen, it is probably Eosin.
I counter-stain the Luxol with:

(1) 10 minutes in Gill 3 Hematoxylin
(2) 3 minutes in Eosin Y Alcoholic.

So I end with a triple stain, an LHE (Luxol Fast Blue-Hematoxylin-Eosin)

The PAS should also work nicely once you differentiate the Luxol.
But as I hardly ever do this, I am  not sure of the protocol.
However Sheehan's "Theory and Practice of Histotechnology" should have a 
Luxol-PAS recipe.

Hopes this helps.
I will send the entire protocol and an image to your email.

Tim Wheelock
Harvard Brain Tissue Resource Center
McLean Hospital

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