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.

Although phospho-specific primary antibodies used in immunohistochemistry (IHC) are expected to detect phosphorylated proteins, in some cases these antibodies may also cross-react with nonphosphorylated proteins. Therefore, it is of ultimate importance to employ a control to determine that the staining pattern is specific. One of the frequently used controls in IHC is a so-called absorption control: phospho-specific primary antibodies are first incubated with a phospho-peptide immunogen to block antibody-binding sites, and this mixture is subsequently applied to tissue sections. If the antibody blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific, but if staining is obtained, the antibody is considered nonspecific. Unfortunately, bound peptide can dissociate from the antibody allowing unblocked antibody to bind to tissue targets, producing unwanted staining. We have developed a simple absorption-control protocol allowing for the efficient neutralization of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of antibody-peptide complex from the incubation solution, minimizing the risk of formation of unblocked antibodies capable of producing tissue staining.

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A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.