I have 5 primary antibodies for immunofluorescence (IF) Staining of tissues. For that, I have only FITC conjugated secondry antibodies. If I'll do IF for 5 different targets but all with same secondary antibodies (FITC), will that create issue in publishing? Will the editor/reviewers ask that it's confusing if the authors have really used 5 different antibodies for IF or they just used one type of anitbody Staining, and they are just showing different parts of same tissue to be considered as 5 different types of antibody IF Staining!!!
If you mean that you will be staining different samples and each sample with only 1 primary ab and your fitc secondary, that is fine. A reviewer should not ask you to redo all that staining but using a different color secondary. For completion, you could run ab controls and take some images just to have and if a reviewer asks them you can share them.
However if you are trying to say if you are 'allowed' to stain 1 sample with all 5 of your primary abs at the same time and then label them with only a FITC secondary then that is of course a big no no.
Hi Jagtar Singh
No, you cannot use 5 primary antibody's together for ICC or IF.
* Using 5 antibodies will cause the Spectral overlap which would make it difficult to distinguish between the signals from different antibodies in confocal or fluorescence microscope. As the secondary antibodies should all have different fluorochromes such as Alexa 488, 594, 647.
* Also you need to carefully consider the cross reactivity and specificity of each antibody you are using. All the five antibodies should have a different source (anti-rabbit, anti-rat, anti-mouse) but specificity towards the same tissues (ex: mice lung/ brain etc.)
So, you can use 2 antibodies at once which is usually advisable or a maximum of 3 (which needs optimization).
Hope it helps,
Thanks,