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Double-Labeling Immunofluorescence: An Alternative to Detect Multiple Antigens in Tissue Sections
I would like to make a comment about the review article: Technical aspects of immunohistochemistry by J. A. Ramos-Vara published in the July 2005 volume of Veterinary Pathology.
In the paragraph: Detection of multiple antigens in a tissue section, the author describes enzyme-based methods to detect two antigens in tissues. Application of sequential staining method (two primary antibodies raised in the same species) and how to avoid overlapping labeling are also discussed.
As far as double-labeling methods are concerned, I would like to point out that double-labeling immunofluorescence represents a good alternative and reliable technique to detect more than one antigen in both routinely processed paraffin and cryostat tissue sections using commercially available reagents.7,9
In my experience, this methodology is very useful either if the two primary antibodies are raised in different species2,3 or if the two primary antibodies are raised in the same species.1,6 In the former, it is very important that the secondary antibodies are derived from the same species in order to avoid that they recognize one another. In the latter, the use of monovalent Fab fragments is suggested for the following reasons: whole IgG molecules and F(ab')2 fragments of IgG have two antigen binding sites; after binding to its primary antibody, most of the secondary antibodies will still have one open binding site, which can capture the second primary antibody from the same species, and overlapping labeling may occur. To label two different antigens with two antibodies raised in the same host species, monovalent Fab fragments of secondary antibodies may be used to sterically cover the surface of the first primary antibody, thus eliminating the possibility of its linking the primary antibody from the second step.4,5,8,10 Of course, the monovalent Fab fragment and the antibody used in the indirect labeling of the second antigen should be labeled with two different fluorochromes. Therefore, in both cases (primary antibodies in the same or different species), the double-labeling immunofluorescence represents a rapid two-stage useful technique.
Although double-labeling immunoenzymatic techniques have been widely used, they are time consuming and very prone to false-positive staining. In addition, according to the author, their use to detect different antigens in the same cell type is not suitable.
Conversely, double-labeling immunofluorescence is quicker to perform; is a very feasible technique also for routine diagnostic laboratory methods7(personal observation) and is the method of choice by using monovalent Fab fragments, to detect two antigens that are expected to colocalize in the same cell type
In conclusion, I think that the importance of double-labeling immunofluorescence cannot be underestimated in the wide field of applied diagnostic and research immunohistochemistry and a citation to it in the review article by J. A. Ramos-Vara was warranted.
Universitá di Napoli Federico II, Napoli, Italia
References
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