Re: Luxol Fast Blue w/ Cresyl Violet

From:Lee & Peggy Wenk <>

Couple of suggestions that work on human tissue, but should also
work on animal:

1. LFB, 55-60 degrees C., for 1-2 hours. Not overnight.
Since the LFB needs to be differentiated out anyways, no
need to do it overnight. It will also now take less time to
differentiate out in the alcohol and lithium carbonate. Plus,
you have just shortened the turn around time!

2. Go back and further, every 10-30 sec., between the 70%
alcohol and the lithium carbonate, until the individual strands of
myelin can be seen, and the background is a VERY pale baby
blue. This is not something that a specific time in differentiating
solutions can be used for. The slides need to be checked
with the microscope. The background will never be clear,
but there should only be a small blush of blue in the gray
matter areas (if blushes can be blue), and individual strands
of myelin are clearly seen. Agitating the slides individually,
up and down, and back and forth in the coplin jar helps.

3. Add some acetic acid to the cresyl echt violet (also known
as cresyl violet acetate). Make the solution about pH 2.5. This
will make it more specific for Nissl substances and RNA, and
less specific for DNA (though the DNA will still stain). So it
will not be so purple everywhere.

4. To the alcohol after the CEV, try adding 1-2 drops of
acetic acid. This again will pull the CEV out of the
background, making it more specific for Nissl. This is
another one in which checking with the microscope will
help. It usually only takes a few seconds. We usually go
from CEV to 95% alcohol with the acetic acid, then to
100%, then xylene. Don't know how well it differentiates
out in 70% alcohol.

Since 70% alcohol is used after the LFB to differentiate
out the LFB, it makes sense that leaving the slide in 70%
alcohol for 6 minutes after the CEV would further
differentiate out the LFB. So we only differentiate in
the 95% alcohol-acetic acid for 2-10 seconds.

Try these ideas, and see how they work on rat tissue.

Peggy A. Wenk, HTL(ASCP)
William Beaumont Hospital
Royal Oak, MI

(Another technical response, Russ!)

----- Original Message -----
From: "Rachel Munshower" <>
To: "HistoNet Server" <>
Sent: Thursday, August 02, 2001 8:31 PM
Subject: Luxol Fast Blue w/ Cresyl Violet

> Hi there!
>    I'm trying to stain rat brains for myelin.  My protocol for this stain
> is LFB overnigh @ 55+ C, 70%EtOH,  Li2CO3 for a few seconds, 2X70% for 1
> min each, water, Cres. Viol for 5 min, 70% if needed, then to 100%,
> coverslip.  My problem is -- too much purple!
>    The first time I did this stain, I got detained and left my LFB slides
> in 70% EtOH for 6 minutes instead of a combined 2, and all my LFB leached
> out.  This time, I watched them like a hawk, and used the final ethanol
> step (about 1 minute) to differentiate the Cresyl violet.  It is still
> HIGHLY purple and the LFB looks dark/royal blue instead of the blue/green
> that I think I am hoping to get.
>    I'm a little bit new at this game, and am not very good at subjectively
> checking slides part way through a procedure to see if they look about
> right.  Our scope is pretty old and it takes me a couple of minutes to get
> a good look and all the while I freaking out because I don't want the
> slides to air dry.  ANYWHO, any recommendations?  I'm afraid to
> differentiate the Cresyl Violet for a really long time because I don't
> to lose the LFB.  I'm considering shortening my Cres Viol time to 3
> minutes. . .
> Sorry this is so long and tedious!
> Thanks,
> Rachel
> ________________________________________
> Rachel Munshower
> IU School of Medicine
> Dept.

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