[Histonet] TTF-1/background staining
Paula Lucas
plucas <@t> biopath.org
Fri Feb 4 08:21:04 CST 2011
Hugh thanks for your reply and I'll type my answers in blue below after the
questions:
_____
From: Hugh Luk [mailto:hlukey <@t> msn.com]
Sent: Thursday, February 03, 2011 6:21 PM
To: plucas <@t> biopath.org
Subject: RE: [Histonet] TTF-1/background staining
Paula,
Incomplete IHC staining has many factors.
1) What type of antigen retrieval are you using? We use the Lab Vision's
Citrate Buffer Ph 6 Can you use a high pH solution instead? Unmasking hard
epitopes (TTF-1 can be stubborn) will look better under a higher pH AR (or
TR for Dako users). Are you re-using your solution? Please say "No" "NO"
or at least "not often." Lastly how are your retrieving? We use Biocare's
Decloaking Chamber, basically a pressure cooker Pressure HIER or rolling
boil methods work best for TTF1.
2) What type of buffer are you using. We use TBS with Tween added: a small
capful to a 5 liter container TBS or PBS should both work. Is it pH'd
correctly? Since you automate, does your buffer contain Tween 20? Too much
or too little could ruin your day.
3) Primary antibody. Concentrate? It's a predilute Could your diluent
(solution) be faulty or your titre incorrect? Call or email Cell Marque to
help you with this? I did call Cell Marque and they gave me some suggestions
such as using an EDTA based retrieval solution and to reduce the antibody
time to 15 minutes, and to also increase the blocker from 5 to 10 minutes
Another email from Tony asks about dilution too, and hints at problems with
the anitbody (AR). I use Dako's version of TTF-1 for human tissues, and I
have not seen a problem. Perhaps you could call Dako and procure a free
sample? I'll try that
4) Fixation. TTF-1 isn't known to be fixation-dependent, but how long are
your tissues sitting in formalin before processing? It depends.we receive
samples from the hospital, and so it can vary. The smaller samples are in
formalin at least 6 hours: includes the sample sitting in the formalin
container waiting for gross, plus then the processor time Problems with over
or under fixation could cause your description. Also, what fixative are you
using? We use 10% Formalin I have heard various fixatives being used on
liver biopsies: Carnoy's, Helly's, Hollende's, Zinc formalin, ect. If you
are using another fixative, can you go back to 10% neutral buffered
formalin?
5) Tissue processor. How is your tissue processor? It's a VIP and the
paraffin is checked at 60 degrees Could your paraffin be overheating?
6) Detection system. I have simply never tried your kit, but I will say
several antibodies from Thermo have worked well. Thermo lists their polymer
system as non-avidin-biotin based, and should be ultra sensitive. If it
works on everything else (especially stains that visualize the nucleus), I
would say your kit is not the problem. But it still might not like your
antibody. For example, the nuclear epitope is rather small and Dako's
polymer kit (Envision plus) was a large bio-synthesized molecule that many
people thought would sterically hinder a good percentage of binding,
reducing the signal. Don't know if that's true, it works okay for us.
7) 30 minute primary incubation should be fine. Is there any chance your
slides are drying while incubating? I'm not 100% sure..the slides look to me
like they are not drying out. We apply the buffer as soon as we place the
slides on the machine, and then we keep the cover to the machine closed
while it's running, so I'm assuming the slides aren't drying out We
sometimes put paper towels on the bottom of the stainer to assure the
chamber is not drying out our slides (but that's only when we get REALLY
paranoid).
8) Liver. Is dirty. Debbie has a point that the liver may be working
against you. You could try a protein block, or something that Thermo may
suggest to deter background. Do you ink your liver biopsies? No we don't
ink the liver bx's Davidson's inks used to give us some problems with IHC on
small tissues (so we went to marking with eosin or hematoxylin).
9) Liver does not normally stain TTF-1. HCC's should be negative. Lung
would stain well (unless your "Lung" tissue is also a metastisis), but for
example breast mets could be focal and weak to diffuse and strong. It
depends on the tumor. Could the tumor be of this type? I will ask the
pathologist this question
Good luck and feel free to email me if something I wrote needs to be
clarified. I apologize for the rambling on... don't apologize! I really
appreciate this
Hugh
Cancer Research center of Hawaii
> From: DSiena <@t> statlab.com
> To: plucas <@t> biopath.org; histonet <@t> lists.utsouthwestern.edu
> Date: Thu, 3 Feb 2011 16:02:22 -0600
> Subject: RE: [Histonet] TTF-1/background staining
> CC:
>
> Hi Paula,
>
> I am probably reading between the lines here but on your TTF protocol do
you use a streptavidin biotin detection system and if so do you block for
endogenous biotin, liver will have endogenous biotin where lung may not have
as much? thanks
>
> Debbie Siena HT(ASCP)QIHC
> Technical Manager | StatLab Medical Products
> Direct: 972-436-1010 x229
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Paula Lucas
> Sent: Thursday, February 03, 2011 2:38 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] TTF-1/background staining
>
> How can I eliminate the background or as you say, non-specific staining?
>
>
>
> I'm no expert here, I admit, and so I'm asking for your help and
> suggestions. I have searched the Histonet archives, and a lot of what I'm
> seeing deals with animal or rodent tissues, and a lot of it was confusing
to
> me.
>
>
>
> To give you a little background info:
>
> We use the Lab Vision stainer and the TTF-1 antibody we use is from Cell
> Marque. We were having issues with this marker from Lab Vision, so we
> switched to TTF-1 a while ago. We use the UltraVision LP detection kit
from
> Lab Vision. It's a polymer driven detection kit.
>
>
>
> The antibody is a ready to use, and we have the time set at 30 minutes.
>
>
>
> We were getting the background staining on a liver specimen last week, and
> we also had this problem today on a lung case. My doctor is getting
> frustrated, and wants me to do something about this, and to repeat the
TTF-1
> stain on the lung, so if someone can give me some suggestions to try, I'm
> ready to try them.
>
>
>
> Thanks in advance,
>
> Paula
>
> Lab Manager
>
> Bio-Path Medical Group
>
> Fountain Valley, CA
>
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