BrdU, is it false positive

[maggieren]

Hi,
I'm trying to do BrdU stainning in rat brain. When I use HCl treatment only, it's hard to detect BrdU signal, so I use HCl denaturing followed by Citrate buffer at ~95C for 15 minutes. The BrdU signal is then easily detected, but all of the sections, including the control one that was not incubated with primary ab are BrdU positive and dark background. I think it could be a false positive result.
Does anybody has good suggestions?
Thank you.

I'd like to attach my picture of BrdU stainning here so that you can help me better, but i don't know how. If anybody want it, I'll send it to your e-mail box. Thanks.

My process is as follows:
dewax slides
denature DNA: 2N HCl for 30min
block endogenous peroxdase:3%H2O2 for 30min
retrieval: 0.01M Citrate buffer at ~95C for 15 minutes
permeabilization: 0.5%Triton X-100 for 20 min
block: 2%goast serum for 30 min
primary ab: 1:500(sigma) 4C overnight
secondary ab: 37C 1h

people succed in BrdU stainning please give me your suggestions, any suggestion, thanks.

[richard03]

I suggest you use citrate buffer AR only and see what happens.

To post pictures, Click here http://www.ihcworld.com/imagegallery/

Then register, check your email to activate your account. Then go to gallery again, login, upload your pictures following the instruction. If you have any problem, please let me know.

Richard

[maggieren]

hi, richard
I found that it was the nonspecific binding of the secondary antibody that caused the false positive result.
However, such binding is not observed on section that was not pretreated with citrate.
Is it possible and how to avoid it?

[hardrob]

i do my brdu staining without any heat pretreatment, since i recognized there is more background staining. my protocol( and it's also the protocol of the cepa/ripa group in europe)
dewax
4N HCl- 30 min
Protease (from Strepomyceus- Sigma) (100mg/100ml PBS Buffer)- 2 min. at 37°C
Rinse in Aqua dest.

Blocking (in order to your protocol)
Staining

i stain all organs with this protocol and it works perfectly, there's one exception: organs that where decalcified  (than i have to check the HCl time)

greets

[maggieren]

thank you, hardrob
i'll try your protocol. but is it 2 or 20 minutes for protease incubation? i ask cause the protocol within the product information to the primary antibody suggests a 30 min incubation with pepsin.
thank you again and i'll let you know what happen.

[hardrob]

no joke, it's 2 minutes. in my opinion it would work without protease. but i have to count the positive cells by image analyzer (to generate a labeling index) and for this reason the protease makes my nuclei-staining........ i don't know how to say.................... ,,evenly distributed stained'' and a bit more intense. so the analyzer detects the labeled cells easier. i take protease instead of pepsin because pepsin causes to mage damage to the morphology and the hematoxilin- counterstain( in my opinion).

greets

[maggieren]

my result is as below: sections without primary ab are almost the same as sections with both primary ab and secondary ab that many dark stained, elliptic cells were observed at the SVZ; but no such cells at SVZ of sections without BrdU injection.

The primary antibody, monodlonal anti-BrdU, is purified mouse immunoglobulin, and the secondary antibody is anti-mouse IgG. The tissue is rat brain.  So is it possible that the secondary antibody bind to the tissue directly after a citrate buffer AR, because of a high homology between rat and mouse?

the above hypothesis is kind of ridiculous, but    is there any reasonable explanation to such strange results?

thank you all for your suggestions.

[maggieren]

hi,
first of all, thank you, hardrob, for your help.
i used pc12 cells to do BrdU staining, without heat pretreatment and it works well, beautiful indeed, indicating that the antibodies and reagents i used are good.
but when it comes to the paraffin sections, problems come again, as i mentioned above.
why and how to resolve it?
any suggestion will be appreciate, thank you all.

maggie

[richard03]

Maggie,

I suggest you change your secondary antibody, i.e. use different species. You may also try other proliferation marker, for example, PCNA, and compare the difference.

http://antibodybeyond.com/reviews/proliferation-marker.htm

Let us know.

[maggieren]

Thank you, Richard.
I'm also considering doing PCNA staining. However, many literatures concerning the proliferation of neuronal stem cells use BrdU staining other than PCNA. I think there must be a reason.
Anyway, thanks and I'll let you know what happens.

[rmcgandara]

Regarding PCNA and BrdU:
"The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous." (PMID: 7678022)

Regarding BrdU staining: I am using the protocol below to stain paraffin embeded sections from mice gut. As you can see I am using a HRP secondary AB instead of and amplified system such as biotin-avidin, but the protocol could be changed to use such systems. I make my own DAB, but you can use a DAB kit. Also I am using Thionin CounterStaining instead of Haematoxylin (it is a personal choice).

Bromodeoxyuridine Staining Using the Boric Acid Buffer Method
Oxford Biotech rat monoclonal anti-BrdU, clone BU1/75 (ICR1)

1.Dewax slides in xylene
2.Air dry and mark with PAP pen
3.Wash in absolute alcohol 3x 5 min
4.Block endogenous peroxidase activity with 1% H2O2 for 30 min
(6.6 ml H2O2 in 200 ml methanol)
5.Place slides in 1M HCL at 60ºC for 8 min.
6.Neutralise slides in Boric acid buffer for 6 minutes
7.Wash in PBS 3x 10 minutes
8.Block with normal rabbit serum 1/20 for 30 min
9.Tap off serum and add rat-anti-BrdU 1/5 for 1 hour
10.Wash in PBS 3x 10 min
11.Add rabbit-anti-rat peroxidase 1/100 + 10% normal mouse serum for 1 hour
12.Wash in PBS 3x 10 min
13.Immerse slides in DAB for 6 min (1 bottle DAB, 200 µl H2O2, 200 ml PBS)
14.Rinse in distilled water
15.80% methanol for 10 min
16.Thionin 6 min
17.95% alcohol 5 dips, 95% alcohol 5 dips, absolute alcohol for 1 min
18.Xylene for 10 min
19.Mount


Hope it helps

[ImmunoNYC]

PCNA is not an effective marker of cell cycle at all. It is expressed IN ALL STAGES OF THE CELL CYCLE INCLUDING G0 and also can be turned on and off in response to DNA repair. Be careful with this one folks.

Thank you, Richard.
I'm also considering doing PCNA staining. However, many literatures concerning the proliferation of neuronal stem cells use BrdU staining other than PCNA. I think there must be a reason.
Anyway, thanks and I'll let you know what happens.

[hardrob]

i agree with MaximinaNYC.
in case of pcna you have to take care for the pretreatment. slight varying in cooking times for example, changes the number of ,,countable'' cells.
in case of brdu it is a ,,yes/no'' thing during evaluation. with pcna you have to be very experienced to say ,,slight coloured nucleus''- ok it's a g1 phase, ,,more intense staining- could be a s-phase''and so on.

by the way: how did you administer the brdu( and how long).

[maggieren]

To hardrob:
BrdU, 50mg/kg in saline, was given through intraperitoneal injection twice daily on 3  consecutive days.

[rmcgandara]

I am using a single i.p. injection of 50mg/Kg 40 min b4 sacrifice and works well.

So I will think that yours should work ok aswell.

Ricardo