Most of us are delighted when we get the dark/royal blue instead of the
greeny blue!  Your problem is probably under differentiation.  When diffing
the LFB diff till you think "Is that enough" then go a little further.  If
you don,t then the Cresyl will accentuate the LFB bringing up that nasty
background.   The tendency then is to blame the Cresyl.  Finallly make sure
you diff the Cresyl  well.  A few drops of acetic acid in the diffing
solution might help.

Throughout the procedure keep agitating the slides and changing solutions.

Andy Shand

----- Original Message -----
From: Rachel Munshower <rachelm@anatomy.iupui.edu>
To: HistoNet Server <histonet@pathology.swmed.edu>
Sent: Friday, August 03, 2001 1:31 AM
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%,
xylene,
> 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
want
> 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. of Anatomy and Cell Biology
> 635