Novel multicolor immunofluorescence technique using primary antibodies raised in the same host species.

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.

Absorption control in immunohistochemistry using phospho-peptides immobilized on magnetic beads.

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.

Modulation of perineuronal nets and parvalbumin with developmental song learning.

Neural circuits and behavior are shaped during developmental phases of maximal plasticity known as sensitive or critical periods. Neural correlates of sensory critical periods have been identified, but their roles remain unclear. Factors that define critical periods in sensorimotor circuits and behavior are not known. Birdsong learning in the zebra finch occurs during a sensitive period similar to that for human speech. We now show that perineuronal nets, which correlate with sensory critical periods, surround parvalbumin-positive neurons in brain areas that are dedicated to singing. The percentage of both total and parvalbumin-positive neurons with perineuronal nets increased with development. In HVC (this acronym is the proper name), a song area important for sensorimotor integration, the percentage of parvalbumin neurons with perineuronal nets correlated with song maturity. Shifting the vocal critical period with tutor song deprivation decreased the percentage of neurons that were parvalbumin positive and the relative staining intensity of both parvalbumin and a component of perineuronal nets. Developmental song learning shares key characteristics with sensory critical periods, suggesting shared underlying mechanisms.