[Histonet] Improving the BrdU staining

Sat Sep 20 12:29:35 CDT 2008

Joost,
You might want to join the NSH IHC Resource Group online at www.ihcrg.org we are a committee thru NSH of over 500 from all over the world working and sharing our expertise in IHC and Molecular Methods.  We have a list serve similar to histonet that is focused just on IHC issues.  Membership with NSH is the only requirement to be part of this resource group.  If you are not a member apply for group membership online anyway and we will tell you how to join NSH.
Best regards,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. #215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
pruegg <@t> ihctech.net
www.ihctech.net
www.ihcrg.org    

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of tf
Sent: Saturday, September 20, 2008 5:12 AM
To: histonet <@t> lists.utsouthwestern.ed
Subject: [Histonet] Improving the BrdU staining

Dear All:

I am writing to discuss the function of each step in BrdU staining.
As you might know, Citrate buffer retrieval at 95 degree for 30 min, and 2N HCl for another 30 min at RT are two critical steps for BrdU staining.

(1) For citrate buffer retrieval: Is this really antigen retrieval? Or we are just using citrate buffer to porize the cell membrane? For antigen retrieval you should slowly cool down the slides in citrate buffer following heating - but I just took those slides out and put them in PBS, and go on with other steps- I can get quite good results. However if I slowly cool down the slides, I found some "dirty spots" between two sections on my slide - do anyone have such experiences?
     I read papers that some people using protease for this step in BrdU staining - I think that's also for porization on cell membrane. Thus, can we just use TBS (Tween or Tris??) to make pores on cell membrane before HCl treatment? What're the differences? 

(2) For HCl treatment: Do anyone has other suggestions on the use of concentration and time for HCl treatment so that the nucleus structure for DAPI staining wont be completely disrupted? The 2N HCl for 30 min at 37 degree really brings very weak DAPI staining then. 

(3) do anyone tried only Citrate buffer step or HCl step? Or you have other topics want to discuss here?

Cheers!

2008-09-13 

tf 

发件人： David A. Wright 
发送时间： 2008-09-12  08:21:47 
收件人： histonet <@t> lists.utsouthwestern.edu 
抄送： 
主题： [Histonet] Re: BrdU 
Hi Joost & the Histonet
I have some general comments on BrdU that i hope help.  Maybe
someone else has more specific ideas relating to your tissue 
too?  
It seems you are having problems with 2 things at once: 
a) switching species
b) switching antibodies
My guess is you only have control over the latter but either
could be the problem! 
a) species - since DNA is DNA, you don't have to worry about
species specificity of your antibody.  Unless you are doing
tissue culture, it's not however always simple to get the same
BrdU incorporation in different species.  I take it that
someone else - possibly different people - labelled the mice
and rats and also that it was done in vivo.  Even repeat
experiments on the same species are variable.  The BrdU
doesn't always go into solution equally (it needs a dilute
base, not buffered saline) and it has to have a phosphate
added on inside the cell before it is incorporated.  So
metabolic rate is important not just dose/kg.  It's a good
idea to collect control tissue from every animal to confirm
incorporation - pick any abundant, proliferating tissue.  I've
often used gut.  You then also have lots of material to work
up and compare different antibodies.  I'm probably not the
only one who'll say you have to try your 2 antibodies side by
side on the same specimen.
b) Different antibodies.  Hmmm.  You mention fixation
procedures but don't say much about what you did to recover
antigenicity in your paraffin sections before staining.  With
BrdU you have to do something just to make it accessible. In
Eukaryotes, (BrdU-containing) nuclear DNA is highly compacted
into nucleosomes with lots of histones and other proteins all
around it and then squashed down a whole lot more.  Fixation
will lock all these proteins together and make it hard for the
Ab to find its target.  I think it's a truism that all nuclear
antigens, including proteins, need some kind of harsh
treatment (heat, strong acid or base, proteinase) to make the
nucleus accessible.  It's interesting that your unconjugated
MAb worked but not the larger conjugated one - maybe it's just
penetration.
On this point, you didn't say whether your protocol was
exactly the same for each Ab.  I've used a bunch of different
anti-BrdU Abs and the manufacturers have quite different
recommendations about pretreatment - 2N HCl, formamide, SSC,
NaOH, proteinase.  If you've only got a tiny bit of tissue,
you MUST do exactly what the manufacturer says [at least the
first time] even if that's different from what worked for you
before with a different Ab (I'd do both!).  It often helps NOT
to block, so the Ab can hit the naked DNA, however exposed.
[c) time.  You mentioned different delays before processing. 
BrdU staining is one of the few things where that won't
matter. As DNA doesn't degrade like proteins, antigenicity
isn't lost with time.] 
Try Again!  Since (same logic) the DNA will still be there on
your slides, IF BrdU was actually incorporated (see a),
there's a very high likelihood that your negative slides WILL
stain with either a different antibody and/or harsher
pretreatment as recommended by the manufacturer.  I liked BD
Bioscience's MAb (clone B44) which only needed a few  minutes
in 0.07N NaOH to unmask to BrdU.  I think it's still available.
Good luck!
-David
--------
David A. Wright PhD
University of Chicago Section of Neurosurgery
---- Original message ----
Histonet Digest, Vol 58, Issue 13 Message: 11
Date: Thu, 11 Sep 2008 12:04:13 +0200
From: "Bruijntjes, J.P. (Joost)" <joost.bruijntjes <@t> tno.nl>
Subject: [Histonet] BrdU
Hi Histonetters
I do have a problem with BrdU, and I hope someone of you can
give a kind of solution/explanation.
I work with NALT (Nose Associated Lymphoid Tissue) of both
rats and mice, which were fixed in a fixative composed of
acetic acid, formaldehyde, ethanol and aqua dest. After 48
hours this fixative was replaced by ethanol. Later on the
tissues were dehydrated and embedded in paraffin. Just because
the lymphoid tissues material is so small, about 10 paraffin
slides were collected.
I started with the rat NALT's. One paraffin slide was stained
with HE, and a few other slides were stained with a monoclonal
antibody directed against BrdU. I was satisfied with the
staining, so far no problem.
After the rats, the same story with the mice NALT's. But I did
not get any positive staining. Some days later I repeated the
first try-out included with some positive controls from the
rat-study with the primary antibody I used in the first part,
and the biotin conjugated primary antibody as well, but again
no positive staining. The pre-treatment of the slides for rat
and mice is the same. The only difference lies in the primary
and secondary antibody.
For rat tissue I use a non-conjugated monoclonal antibody,
followed by HRP-conjugated powervision.
For mice tissue I use a biotin conjugated monoclonal antibody,
followed by a HRP-conjugated streptavidin.
Can anyone give me an explanation? The storage of the slides
was in a room with an equal temperature (about 20-21?C) and
humidity.
Is it possible that the epitopes in the single slides are
destroyed so that they are not recognizable anymore by the
antibody? The time between preparing the slides and the BrdU
staining of the rats NALT slides was at the most 4 weeks,
while the first negative staining on the mice NALT's appeared
after about 8 weeks.
Thanks in advance
Joost Bruijntjes
TNO Quality of Life
Zeist
The Netherlands
TNO.NL <http://www.tno.nl/>
Joost Bruijntjes
T +31 30 694 44 80
F +31 30 694 49 86
E joost.bruijntjes <@t> tno.nl <mailto:joost.bruijntjes <@t> tno.nl>
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026
5841 S. Maryland Ave
Chicago, IL 60637 USA
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Skype: davidanthonywright
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