Re: [Histonet] Luxol Fast Blue tissue damage

From:John Kiernan

You have provided a superb description of ice crystal holes
in CNS tissue. This is due to slow freezing of the specimen,
which must be prevented. An abundance of advice is available
on the Web, and it's all been there in printed books for 
more than 40 years. 

Your staining method includes some crazy steps. Have you looked 
up the method in a book? There are various modifications of the 
original method, and the mechanism of myelin staining by luxol 
dyes is quite well understood. 

Your 'regular large "holes" in my tissue' do not originate
"after staining" for myelin. The empty spaces were formed when
the specimens were too slowly frozen. 

John Kiernan
London, Canada.
Mathew DeGutes wrote:
> Hey all any help is appreciated.  I am doing some myelin staining with luxol fast blue and have
> found far too often that I get regular large "holes" in my tissue after staining.  My basic
> protocol is this:
> -Cryosections on slides rinse in ddH20 then PBS then 70% ethanol
> -Place in .1% Luxol Fast Blue in 95% Ethanol and .05% Acetic Acid over night at 55C
> -Remove from LFB and wash in ddH20 then PBS
> -Differentiate in .05% Lithium Carbonate (usually about 30 sec)
> -Wash in 70% ethanol and then counterstain or dehydrate and coverslip
> The actual staining looks pretty good, but I just get these Round white holes as if something
> bubbled up there and destroyed the tissue.  Nothing is boiling so I am curious if anyone else has
> run into this problem and what might ameliorate it.  Thanks again.
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