Author Topic: Double IHC...double antigen retrieval?  (Read 7385 times)

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Offline tyler

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Double IHC...double antigen retrieval?
« on: March 13, 2008, 09:40:11 AM »
Hi everyone! I'm new on the forum and I have a problem I need your help to solve
I need to make a double IHC, one against a membrane antigen and one against a nuclear antigen

I have three different problems:
1) do I have to use different antigen retrieval techniques? one antigen is unmasked via citrate buffer and heat, while the other one is unmasked with citric acid and heat...
2) if I need to do both, won't heat disrupt my first signal?
3) to reach the nuclear antigen, I'd usually use triton...won't it disrupt my membrane signal?

thank you very much, I really need your help!

Double IHC...double antigen retrieval?
« on: March 13, 2008, 09:40:11 AM »

Offline richard03

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Re: Double IHC...double antigen retrieval?
« Reply #1 on: March 13, 2008, 08:01:09 PM »
1) do I have to use different antigen retrieval techniques? one antigen is unmasked via citrate buffer and heat, while the other one is unmasked with citric acid and heat...

Citrate buffer and citric acid are similar so try both antibodies using citrate buffer and it most likely will work.

2) if I need to do both, won't heat disrupt my first signal?

If 1) worked, there is no problem.

3) to reach the nuclear antigen, I'd usually use triton...won't it disrupt my membrane signal?

Use low concentration triton would not disrupt the membrane staining. Even high concentration will still work at Light Microscopy level.

Offline tyler

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Re: Double IHC...double antigen retrieval?
« Reply #2 on: March 18, 2008, 09:02:03 AM »
Thank you! I used just citrate buffer and no triton, and it did work :)
thank you for your help!

Re: Double IHC...double antigen retrieval?
« Reply #2 on: March 18, 2008, 09:02:03 AM »