[Histonet] Luxol Fast Blue with IHC

Hofecker, Jennifer L jennifer.l.hofecker <@t> Vanderbilt.Edu
Wed Aug 26 14:25:15 CDT 2009

Hi Nicole, 
I do LFB with IHC on FFPE human brain but there are a few differences in
our protocols. I have stained LFB with Neurofilament and with S-100 but
never with Ki-67. I also use DAB as my chromagen and differentiate the
LFB with dilute Lithium Carbonate.
I'm not familiar with the VIP chromagen you mention, so I'm just
guessing here (Perhaps somebody on histonet can fill in that blank for
us). If VIP chromagen is insoluble in alcohol/xylene, you will likely be
fine. I do reduce my staining time in LFB a little so that it doesn't
overpower the IHC. You may want to try an LBF protocol with lithium
carbonate. It won't hurt IHC at all. One other hint: LFB before IHC
doesn't work for me. When I perform antigen retrieval, all of my LFB is
lost. If you haven't tried it yet with your current reagents, that would
be my first step. If you KNOW that VIP will be removed, I'd try to
"borrow" some DAB from a neighbor.

Good luck and don't hesitate to shoot me an email if you have any
additional questions.

Jennifer L. Hofecker HT(ASCP)
Vanderbilt University Medical Center
Division of Neuropathology
Nashville, TN 
ph 615.343.0083
fax 615.343.7089

-----Original Message-----
From: Patten, Nicole (NIH/NIAAA) [F] [mailto:pattennj <@t> mail.nih.gov] 
Sent: Wednesday, August 26, 2009 10:03 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Luxol Fast Blue with IHC


Has anyone tried to counterstain an IHC stain with Luxol Fast Blue? I am
worried that the LFB destain will remove some of the IHC. 

I use the VIP solution from Vector as the chromogen for IHC instead of
DAB and I destain Luxol Fast Blue with Hydroquinone/Sodium Sulfite (in
addition to the ethanol steps). I'm staining for Ki-67 and I'm doing
this on FFPE human brain sections. I haven't found anything in the
Histonet archives on this topic.

Any advice would be greatly appreciated. Thanks in advance!

-Nicole, Post-bacccalaureate Fellow, NIH

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