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

From:"Bonner, Janet"

We do the LFB/PAS by doing the LFB part of the stain and counterstaining with PAS. I'm not sure about the digestion as this could radically change the proceding reactions.
from: on behalf of John A. Kiernan
Sent: Tue 4/25/2006 1:15 PM
Subject: Re: [Histonet] LOOKINF FOR LUXOL FAST BLUE/PAS Staining Procedure

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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