From:Tim Wheelock

Hi Everyone:

Lately, I have been having problems with my Luxol Fast Blue myelin stain 
coming out too pale.
I have gone through each step and can't figure out where the problem 
I even bought another bottle of the dye itself to no avail.

I rechecked and re-made the The Luxol Fast Blue working solution (0.1 
gram Luxol Fast Blue and 1 ml of 10% glacial acetic acid in 100 mls of 
95% ethanol).
I did the same for the differentiator(1% Hydroquinone/5%Sodium Sulfite).
That made no difference at all.
The sections look pale after I remove the excess stain in 95% before the 
differentiator, so I assume it is not a mistake making up the later or a 
problem with the H+ E that I add onto the myelin stain.

I adjusted by increasing the incubation time in a 60C oven from 2 to 4 
And decreased the time in the differentiator from 2 dips to 1.
This helped a lot, but I knew that these changes did not get to the root 
of the problem.
Yesterday, they still came out pale even with these changes in place

I keep thinking the the problem is in the working solution but am not 
sure how.

(Fixation, processing and sectioning are the same as they have always been.)

I have done this stain for years and never had a problem...until now.

Thanks for any suggestions you may have.

Tim Wheelock
Harvard Brain Tissue Resource Center
203 Mailman Research Center
McLean Hospital
Belmont MA 02478
Phone: 617-855-3592
Fax: 617-855-3199

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