On Wed, 28 Mar 2001, Andrea Grantham wrote:

> Everybody mentions LFB-ORO in their discussion but I don't have a real 
> procedure. What I need to know is this, do you do the LFB and then ...

> Another method talks of Neutral Red to include Nissl. I have Neutral Red 
> Iodide. Is this what I want to use? If not I'll need to get plain old 
> Neutral Red ...

Andi, I'll be happy to send you some technical instructions, but
your emails clearly show that your lab's greatest need is for half
a dozen textbooks, and perhaps also a principal investigator who
has spent a few hours reading from one or two of them.

The LFB and ORO method has to be a tricky one because LFB must
be used in alcoholic solution, and this dissolves both ORO and
the hydrophobic lipids in which ORO dissolves. In my earlier
email I said I hadn't tried this method. The reason is because
it seems contrary to the simple logic of what mixes with or
dissolves in what, and there's no reason to expect this method
to work well. I suspect that impressive results are
occasionally seen but cannot be predictably reproduced.

This is a field where thinking about mechanisms of staining
is paramount. Someone suggested adding neutral red as a Nissl
stain. That's fine after LFB alone, but it's the same colour
as ORO!  Moreover, neutral red (or any other basic dye) will
bleed out into almost any aqueous mounting medium, which is
necessary for retaining ORO. (An alkaline medium would retain
neutral red, but it would also extract luxol fast blue.) 

Any staining strategy that combines different mechanisms of
coloration needs to be carefully thought out, starting with
the principles that govern the way each part of the procedure
works. Bench experiments are also needed (always) to determine
things like the timing of the several steps in the method.

----------------------------------------
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1
   kiernan@uwo.ca
   http://publish.uwo.ca/~jkiernan