Re: [Histonet] LOOKINF FOR LUXOL FAST BLUE/PAS Staining Procedure

From:"John A. Kiernan"

Nobody seems to have answered this. Assuming that
LFB is luxol fast blue for myelin, the following
is based on the procedure of Kluver & Barrera
(1953) J. Neuropath. Exp. Neurol. 12:400-403.
There are other variants; this is the only one
I've used myself.

Solutions required:

Staining solution.

Luxol fast blue MBS:  0.25 g
95% ethanol:           250 ml

Many workers add glacial acetic acid (2.5 ml) but
this does not appear to confer any advantage. The
solution keeps for at least 5 years and may be
used repeatedly.

Differentiating solution (0.05% Li2CO3).

Lithium carbonate (Li2CO3):  0.25 g
Water:                        500 ml

This keeps indefinitely and can be used
repeatedly, but the solution should be discarded
when it is more than faintly blue from extracted

Procedure for formaldehyde-fixed tissue:

1. De-wax paraffin sections and take to 100%
ethanol. Frozen sections should be dried onto
slides (from water) and then equilibrated with
100% ethanol.
2. Stain in luxol fast blue solution in a
screw-capped staining jar at 55-60C (in an oven or
water bath) for 16-24 hours (overnight).
3. Rinse in 70% ethanol and then in water.
4. Immerse in the differentiating solution until
grey and white matter can be distinguished. This
commonly takes 2 to 30 seconds for thin paraffin
sections, but thick frozen sections may require up
to 30 minutes. Move to Step 5 early rather than
late, to avoid extracting too much blue dye.
5. Transfer slides to 70% ethanol, two changes,
each 1 minute. More dye leaves the sections at
this stage.
6. Rinse in water. Check with a hand-lens or low
power microscope. Grey matter should be largely
unstained; white matter should be a greenish shade
of blue. Carefully repeat Steps 4 and 5 if there
is still blue colour in neuronal cell bodies in
grey matter.
7. Counterstain with an aqueous solution of a
contrasting cationic dye at pH 3.5 to 4. (0.5%
neutral red, CI 50040, is good) to show Nissl
substance of neurons and nuclei of all cells. 
8. Wash in water and blot dry.
9. If necessary, differentiate the counterstain in
70% ethanol until only the nuclei and Nissl
substance are stained. There should be no
"background" colour in the neuropil between
neuronal cell bodies in grey matter. The colour of
the stained myelin is deepened by counterstaining.
10. Blot dry and dehydrate in three changes of
100% ethanol (each 30 seconds with agitation) or
n-butanol (each 3-5 minutes, without agitation).
(Use n-butanol if you find that ethanol extracts
too much of the counterstain.)
11. Clear in xylene and cover, using a resinous


Myelin blue; nuclei and Nissl substance the colour
of the counterstain.


1. Some batches of luxol fast blue MBS do not work
very well. For other techniques, see Cook (1974).
Churukian (1997) describes a variant in which the
staining is accelerated by heating in a microwave
oven. Luxol fast blue ARN can be used for myelin
staining, with minor variations in technique.
2. Neutral red is an excellent counterstain for
luxol fast blue. Another counterstain that is
sometimes used is the PAS procedure which provides
a pink background in the neuropil, without nuclear
or other cellular staining. This seems a rather
pointless thing to do.
3. For the mechanism of staining, see R.A.Clasen
et al. (1973) J. Neuropath. Exp. Neurol. 32:
4. I don't know why this method is still used.
Myelin staining with a ferric salt and eriochrome
cyanine R (= chromoxane cyanine R, = solochrome
cyanine R, = CI Mordant blue 3, CI 43820) has been
around since 1965, uses a much less expensive dye
whose chemical identity is known, takes only 30
minutes, and gives a stronger blue colour to
myelin than luxol fast blue. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
_______________________________ wrote:
> Cannot locate an exact staining procedure for LFB/PASH.  Is anyone currently using this stain and if so can you forward me your procedure.
> Thanks
> _______________________________________________
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