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Author Topic: blocking  (Read 3343 times)

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Offline weneng

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blocking
« on: May 27, 2005, 02:29:10 PM »
Hello,
I need your advice on blocking. The animal was injected with human antibody.  After the tissue are havested I have to do IHC using the same human antibody.  I know the secondary antibody will pick up the antibody that is injected as well as the one I applied as primary antibody in IHC. I hope I said it clearly.  So please tell me what kind of blocking I can use to block the injected human antibody? And how? Will normal human serum work?

Any input will be highly appreciated!

Wen

blocking
« on: May 27, 2005, 02:29:10 PM »

Offline broiler25

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blocking
« Reply #1 on: May 27, 2005, 03:27:03 PM »
Why are you using the same antibody for two different applications in the same animal?  Instead of adding the same primary antibody a second time, why don't you try to detect the injected antibody by just using an anti-human secondary?


If you're really intent on doing this- here's how you do it.  Use an anti-human monovalent F(ab) before you do your IHC stain.  Make sure it is raised in the same species as your secondary antibody to prevent cross-reactivity.     Using this technique, the epitopes of first primary antibody are saturated.     Go to jacksonimmuno.com for more detailed info.  I use this technique and find it to be quite effective when using two primary antibodies.

Offline weneng

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blocking
« Reply #2 on: May 27, 2005, 05:35:13 PM »
Thanks!
But my method is that I need use three antibodies: human primary, rabbit anti-human secondary and Envision anti-rabbit. So I couldn't use rabbit anti-human IgG to block. Any suggestion please?

Wen

Offline richard03

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blocking
« Reply #3 on: May 27, 2005, 11:32:21 PM »
What about using "goat anti-human monovalent F(ab)" instead of "rabbit anti-human monovalent F(ab)" to do the blocking job?

Richard

Offline ImmunoNYC

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blocking
« Reply #4 on: May 28, 2005, 05:55:58 PM »
Why not biotinylate or FITC label or conjugate to something your antibody for the IHC portion? This WILL work if the conjugation is done properly.

There are several great kits on the market for conjugation to make your life easy.

Offline weneng

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blocking
« Reply #5 on: May 31, 2005, 01:49:56 PM »
Thanks for all suggestions! I am going to try mouse anti-human F(ab), so that will not cause background with my Envision.

I tried all kinds of conjugated ones, biotin, AP and FITC. But none of them worked after many many tests. Maybe the conjugation process is not good enough like you said.

Thanks again!

Wen

blocking
« Reply #5 on: May 31, 2005, 01:49:56 PM »