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Author Topic: re: Double immuno problems  (Read 2767 times)

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Offline LiRasch

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re: Double immuno problems
« on: August 11, 2015, 12:14:26 PM »
Hello,
I am currently carrying our double IHC-P on paraffin-embedded tissue using a combination of:
1. Dako Envision double staining (mouse/ rabbit, HRP/AP)
2. Dako EnVision single stain (mouse, HRP) and Biotin-Avidin-AP (rabbit, AP).

I have tried several variations of method (1), however, I am experiencing significant problems getting primary antibody 2 to react. Primary one reacts just fine, however, I never get a signal from Ab2 following the DAB colour reaction.

Conversely, method (2) works well adding both primary Ab1/2 as a cocktail, followed by both secondaries, and develop both in immediate succession at the end of the process.
This method is therefore fine when using a combination of mouse/rabbit secondaries, however, when using two anti-mouse secondaries (most of the antibodies available to me), this is not feasible due to cross-reactivity issues.

I therefore need to establish a modified Envision double stain method whereby primary Ab1/ secondary Ab1-HRP set can be coloured reacted, followed by application of Ab2/ secondary Ab2-AP & successful colour reaction.

Even with a second round of antigen retrieval (heat-mediated) following development of Ab1 with DAB, there is no signal from Ab2 (Vector Red).
Any ideas as to what might be preventing the second primary from working would be greatly appreciated!

Liam

re: Double immuno problems
« on: August 11, 2015, 12:14:26 PM »

Offline gula

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Re: re: Double immuno problems
« Reply #1 on: August 11, 2015, 01:16:43 PM »
Is it possible, that the AB1-reaction covers the AB2-reaction? Same localisation?
Have you tried to exchange AB1 and AB2? Same result?

Dako doesn't explain the exact mechanism of their double stain blocking reagens in the working procedure. But it has to have an effect on the bound antibodies. Could there also be an influence on your second antigen?

I would try to omit the blocking reagens and do a HIER-step (5min, 80C) instead. This should be enough to denaturate the bound antibodies.
gula

Offline LiRasch

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Re: re: Double immuno problems
« Reply #2 on: August 11, 2015, 05:34:07 PM »
Hi Gula,

Thanks for responding to my query. I would say co-localisation isn't an issue in this case, as I have ensured a combination of cytoplasmic, extracellular or nuclear markers, so as to avoid signal overlap. Indeed I have exchanged AB1 and AB2, both of which work well when applied as the first antibody. The second antibody produces no discernible signal. I am currently working on the assumption that when the AB1 colour reaction (DAB) is carried out, this somehow re-masks the AG2 rendering AB2 inoperable, even with a second round of HIER. Sure, I could leave the blocking reagents out and see how this works out; I'll give that a go. However, one of the antibodies I am using as a +ve control requires an extended period of HIER, which is why I run this for 40 minutes.

My assumption that the initial DAB reaction re-masks AG2 is drawn from the fact that if I apply both primaries as a cocktail, then both secondaries, and finally colour react them in succession, both produce a signal - however, this only works with primary antibodies raised against different secondaries (e.g. mouse and rabbit), otherwise the application of the conjugated secondaries (e.g. both mouse) will result in cross-reactivitiy.

Complicated stuff!
 

Offline gula

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Re: re: Double immuno problems
« Reply #3 on: August 12, 2015, 05:12:33 AM »
HIER has to be done before the first staining for both antigens. And therefore one should only combine antigens, that need the same pretreatment. Have you taken this into consideration?
The HIER-step inbetween is only necessary for the denaturation of the added antibodies and can be short.

I think it is possible, that DAB can mask the antigen. But you say, that co-location is no issue.

One thing, one should always think about, is the quality of the reagens. Maybe some of the second-part-reagens are not working? New kit?
Gula

Re: re: Double immuno problems
« Reply #3 on: August 12, 2015, 05:12:33 AM »