Hi! You can do it if the host species of the biotinylated antibody and the unlabeled antibody are different. You should use fluorescence conjugated secondary that shows no cross-reaction with the biotinylated antibody. (The best way if the biotinylated and the fluorescence labeled antibodies are from the same species.)

hello dear,

It's possible.........and as antibodies are for different Ag so least cross reactivity is expected but I will suggest to first label cell with Biotinylated ab and then labelling for another marker with FITC conjugated secondary-ab as not doing this can result in sufficient quenching of FITC signal. 

If u want to merge images from both antibodies then this approach can work well. If thts not the issue then u can also try stripping of an antibody after u r done with photography and then labelling with another ab.

Hope it helps..!

agree with the comments above

Hi

Thank you for your comments. I was wondering if there is any protocol for that purpose.

Thanks

please read this article :

http://www.sciencedirect.com/science/article/pii/S0065128105000280

A multicolor fluorescence immunostaining technique for simultaneous antigen targeting

Igor B. Buchwalow, ,

Evgeny A. Minin,

Werner Boecker

 Show more

Summary

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. Here, we briefly outline different approaches intended to close this technological gap and focus on multiple immunolabeling with monoclonal primary antibodies. To this end, we generated a basic universal protocol for the use of secondary antibodies selectively recognizing different isotypes/subclasses of monoclonal primary antibodies. This approach is widely applicable and offers a simple procedure for simultaneously detecting two or more antigens.

Keywords

Immunocytochemistry;

Immunohistology;

Multiple immunolabeling;

Monoclonal primary antibodies

 Metastasis Research Protocols

Methods in Molecular Medicine Volume 57, 2001, pp 41-48

Multiple Labeling Techniques for Fluorescence Microscopy

Amanda J. Atherton, Catherine Clarke

Thank you Karima, I will have a look. the biotinylated antibody is arised from a plant and the host from another primary Ab is rabbit. I hope I could find a good appraoch for doing double labeling for my tissues

Hi:

Why not use two different fluorophores (i.e. Fluorescein and Rhodamine or another) that absorb (excite) and then emit at different wavelengths and then merge the image results.

Dr. Veltri 

Hi Robert

the point is in our lab we already have that biotinylated antibody and we were wondering if we could use that in our experiment along with a fluorescence approach to detect another antigen in the tissue

Hi:

It is very simple: In the first step, you should use an indirect fluorescence approach (primary Ab and fluorescence conjugated secondary Ab) for detecting the first  antigen. 

In the second step, you dare apply the next primary haptenylated (biotinylated) antibody against another antigen. Biotin you can visualize with streptavidin conjugated with a fluorophore of a color different from the color of the fluorophore used in the first step.

It does not matter if the host species of the biotinylated antibody and of the unlabeled antibody are different (as think Laszlo Cervenak and others) or are the same. You find the protocols in the following publications:

Boecker W, Junkers T, Reusch M et al. (2012) Origin and differentiation of breast nipple syringoma. Sci Rep 2:226

Boecker W, Stenman G, Loening T et al. (2013) K5/K14-positive cells contribute to salivary gland-like breast tumors with myoepithelial differentiation. Mod Pathol 26:1086-1100

Boecker W, Stenman G, Loening T et al. (2014) Differentiation and histogenesis of syringomatous tumour of the nipple and low-grade adenosquamous carcinoma: evidence for a common origin. Histopathology 65:9-23

Buchwalow I, Samoilova V, Boecker W et al. (2011) Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Sci Rep 1:28

Buchwalow IB, Boecker W (2010) Immunohistochemistry: Basics and Methods. Springer Heidelberg, Dordrecht, London, New York

Hi! Actually Igor ignores that if the biotinylated antibody is from the same species as the unlabeled one then it can bind to the unoccupied arm of the fluorescence labeled secondary antibody. Therefore, the signal of the biotinylated-antibody/streptavidin-fluorophore will be false, it will show the location of both antigens.

But this is just an academic question since it turned out that the unlabeled and the biotinylated Abs are from different species, so they can be used together. (Anyway, what do you mean plant antibody? Plants do not make antibody unless you use plantibody technology, but in this case there is a real host species from which the Ab genes were transfered to a plant.)

Hi Laszlo

The biotinylated marker which I am talking about is Isolectin GS-IB4 which is isolated from the seeds of the tropical African legume Griffonia simplicifolia. By your comments (you and others) I am hoping to successfully double stain the endothelial cells in bone marrow.

Hi Mohammadhossein! Well, in this case there will be no problem, it does not matter if you do the double labeling consecutively, or simulataneously.

Dear Laszlo!

You speculate that if the biotinylated antibody is from the same species as the unlabeled one, then it supposedly  could bind to the unoccupied arm of the fluorescence labeled secondary antibody.  In fact this theoretical interaction does not take place, and we could prove it.

Boecker W, Stenman G, Loening T et al. (2013) K5/K14-positive cells contribute to salivary gland-like breast tumors with myoepithelial differentiation. Mod Pathol 26:1086-1100

Boecker W, Stenman G, Loening T et al. (2014) Differentiation and histogenesis of syringomatous tumour of the nipple and low-grade adenosquamous carcinoma: evidence for a common origin. Histopathology 65:9-23

Buchwalow IB, Boecker W (2010) Immunohistochemistry: Basics and Methods. Springer Heidelberg, Dordrecht, London, New York

If you manage to prove your purely theoretical speculation and publish it, I will send you a bottle of a good old Scotch.  Please let me know it and then send me your address.

Double, even triple labeling, techniques are widely used. I would stain the way you have proposed but make sure the antibodies are from different species. I see that there is some debate here on whether or not this is necessary, but if you have the resources available I would use antibodies from different species to be safe.