Double immunofluorescent staining using two unconjugated primary antisera raised in the same species

Monoclonal antibodies (MAbs) capable of recognizing developmental stage-specific neuronal epitopes are becoming increasingly available. Because most of these MAbs are raised in a single species (mouse), simultaneous immunofluorescent detection of multiple epitopes has been difficult. We have taken advantage of the high sensitivity of tyramide signal amplification to develop a protocol that permits simultaneous detection of two antibodies raised in the same species. One primary antibody was applied at a concentration below the detection limit of fluorescently labeled secondary antibodies, yet sufficient for detection with the tyramide system. This first primary antibody was then effectively neglected during application of a second primary antibody that was detected by conventional fluorescently labeled secondary antibodies. Specifically, dual labeling for nestin and MAP2 was used to distinguish neuronal stem cells and precursor cells from immature postmitotic neurons, and synapsin I and GAP43 immunostaining was used to distinguish neurons with established synaptic connections from developing neurons. We have used this technique for staining both tissue sections and cultured ails from the embryonic mouse brain. This technique should be widely applicable and offers a simple procedure for simultaneously detecting two antigen when antibodies from only a single species are available.