Author Topic: IHC better than IF for same antibody - why?  (Read 1689 times)

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Offline SJessica

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IHC better than IF for same antibody - why?
« on: July 20, 2015, 10:39:14 AM »
Hi,
I ran IHC using an antibody for A4GNT on a stomach tissue (positive control) and a spleen tissue (negative control) and it works really well!  :)

I ran IF with the same antibody, same dilution, on the same stomach tissue, and it BARELY works.  :-\ Does anyone know why this is?
What could I do to enhance and improve my detection in IF?

Jessica

IHC better than IF for same antibody - why?
« on: July 20, 2015, 10:39:14 AM »

Offline gula

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Re: IHC better than IF for same antibody - why?
« Reply #1 on: July 20, 2015, 03:07:59 PM »
In IHC an enzyme proceeds the substrate-chromogene and it converts a certain amount of chromogen in relation to the bound primary and secondary and in relation to duration of incubation.
In IF the signal only relies on the amount of the bound antibodies that carry the fluorochrome.

So the kind of producing a signal is rather different and it is rather logical, that the antibody-titer can be different.

It depends also on the applied method. one-step, two-step, three-step. Every step leads to a further amplification of the signal.

I would try to prolong the antibody-incubation and to increase the antibody-concentration, until too high background.
good luck, gula

Offline SJessica

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Re: IHC better than IF for same antibody - why?
« Reply #2 on: July 21, 2015, 03:41:26 PM »
Thanks Gula! My protocol for IHC and IF is very similar, except for the DAB use.

Here are my next few questions then:
  • Is a permeabilization step necessary? Clearly the primary Ab binds, as I confirmed through IHC.
  • Someone who had this question earlier was responded to with the solution of trying TBS instead of PBS. Is that really going to make that much of a difference?

Thanks so much for your prompt response :)

Offline gula

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Re: IHC better than IF for same antibody - why?
« Reply #3 on: July 23, 2015, 01:19:30 PM »
Permeabilization increases the sensitivity of your test. If the IF has an overall lower sensitivity permeabilization can help.
What is the processing of the tissue? formalin fixed paraffin embedded; formalin fixed and frozen; only frozen?

For FFPE-material antigen retrieval is helpfull till necessary for a successfull IHC/IF. And even for unfixed tissue it is reported, that a short AR-step improves the results.
I don't think, there is a big difference between PBS and TBS. PBS should be avoided, when working with phosphatase.

gula

Re: IHC better than IF for same antibody - why?
« Reply #3 on: July 23, 2015, 01:19:30 PM »