Re: [Histonet] Luxol Fast Blue

From:Gayle Callis


Fine tuning the luxol fast blue (LFB) can be touchy sometimes.  We use the 
classic Kluver Barrera method

LFB overnight in 60C waterbath,
Brief 95% ethanol rinse to remove LFB
Distilled water
Differentiate in 0.05 to 0.08% lithium carbonate approximately 10 to 20 
dips, then go to 70% and carefully watch the LFB stream away from the 
section.  Beware, this is FAST!!   It is important to NOT dip in 70% 
ethanol too long.  Rinse really well with distilled water (use several 
changes) and use the microscope to see if myelin is well distinguished or 
the white matter from gray matter. If you need a bit more blue removed, 
then repeat the lithium carbonate and 70% alcohol step, very, very carefully.

We have found brain differentiates a bit faster than the spinal cord and 
your sections are a bit thicker although some do 10um sections for 
brain/spinal cord.    You may be overdifferentiating as you want to see the 
myelin (this tends to look like strands of blue tissue).  After rinsing 
thoroughly with distilled water, do the PAS stain - we like 15 min in 1% 
periodic acid then 20 minutes in PAS, rinse with running tap water for 10 
min.  There are other variations on time with PAS.

When you are finished,  start your dehydration in 95% alcohol then 100% and 
do these steps very quickly by dipping.   We never go back to 70% after 
just LFB or PAS.

By purple color, do you mean more of a gross observation of the section as 
this would seem normal..  Microscopically you see the blue myelinated areas 
with reddish pink PAS structures and not a purplish overall color.   When 
we look at our sections macroscopically (no microscope) and with EAE virus 
murine model, we can still see areas of demyelination on edges of cord, but 
the overall color of LFB-PAS is a bit purplish.

  At 10:52 AM 1/12/2007, you wrote:
>I'm trying to stain mouse spinal cord sections, (7 um), with Luxol Fast 
>Blue and periodic acid/Schiff and hematoxylin.  In the literature I've 
>been reading, there are some great examples of a nice pink to red stain of 
>the grey matter, and a nice solid blue stain of the white matter.
>Mine look not so good, (purple),  and I've been struggling for a while 
>with it.  I've tried so many adjustments to the protocol that it would be 
>too long to list here.  But I think the heart of my problem is with LFB, 
>differentiating the grey matter completely with out fading the 
>blue-stained white matter.  Somebody please tell me the secret!
>I've experimented with the LFB alone, and believe that differentiating in 
>lithium carbonate, with or with out 70% EtOH, should clear the grey matter 
>of the stain, and leave the white matter blue or turquoise.  But it only 
>clears it somewhat, and any further attempt to differentiate also quickly 
>fades the white matter.  Any help is greatly appreciated.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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