Novel Multicolor Immunofluorescence Technique Using Primary Antibodies Raised in the Same Host Species

Abstract

Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of each secondary antibody with multiple primary antibodies when doing either dual- or triple-labeling immunofluorescence, it is necessary to use primary antibodies raised in different host species such as mouse, rabbit, and goat. However, in many cases, suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the first target protein followed by detection with antiprimary secondary antibody conjugated to NorthernLights™ NL-637 tag (fluorescence in the far-red spectral region). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-493 tag (green fluorescence) and anti-DIG secondary antibody conjugated to a Rhodamine Red X™ tag (red fluorescence). This technique provides good spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have only primary antibodies raised in the same host species at their disposal.

HDAC8 inhibitor attenuates airway responses to antigen stimulus through synchronously suppressing galectin-3 expression and reducing macrophage-2 polarization

“…Double immunofluoresence staining has been applied for examining distribution of two different antigens in the same tissue which retains excellent morphology [51,52]. Moreover, the color labels for two primary antibodies can be detected simultaneously using an antibody cocktail by probing antigens [53]. Our results not only provide insight into the localization of independent antigens within the tissue and cells but also suggest that HDAC8 and Gal-3 as convergent targets were co-interfered in the antigen-induced lung disease.…”

Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies

“…They also used an FITC-conjugated secondary antibody for the indirect detection, but used streptavidin, instead of avidin, that was conjugated to Texas Red, rather than TRITC. Most recently, Frisch and colleagues (2011) developed a method for performing triple immunofluorescence with primary antibodies from the same species. In addition to combining indirect detection with biotin-streptavidin-mediated direct detection, they added a digoxigenin-mediated indirect detection step to enable detection of a third protein.…”

Novel Synthetic (Poly)Glycerolphosphate-Based Antistaphylococcal Conjugate Vaccine

“…pgp-tetanus toxoid (pgp-TT) conjugate was prepared using the HyNic/4FB conjugation couple (Fig. 1B) (30,31). Specifically, TT was modified with amino-reactive S-HyNic to incorporate aromatic hydrazine groups on the side chain amines of the lysine moieties.…”