Triple immunofluorescence labelling of parvalbumin, calbindin-D28k and calretinin in rat and monkey brain.

This study presents novel techniques for the concomitant cytochemical detection of the calcium-binding proteins parvalbumin, calbindin-D28k and calretinin which are frequently used neuronal markers. For the triple immunofluorescence labelling of such antigens in rat and monkey brain--with emphasis on the cortex--we developed four different protocols which revealed obviously identical distribution patterns in consecutive sections. These methods included the simultaneous use of purified monoclonal antibodies directed against parvalbumin and calbindin--D28k--haptenized with biotin or digoxigenin--and subsequent visualization with fluorochromated hapten-recognizing immunoreagents. For the combined visualization of the calcium-binding proteins we applied the bright red fluorescent carbocyanine Cy3, blue fluorescent 7-amino-4-methylcoumarin-3-acetic acid (AMCA) and as green fluorophore either fluorescein or the newly introduced carbocyanine Cy2. The latter showed a higher fluorescence intensity and more resistance against photobleaching than fluorescein. In addition to clearly distinguished distribution patterns of the calcium-binding proteins, neurons co-expressing parvalbumin and calbindin-D28k in the parietal and piriform cortex of rat were demonstrated. The elaborated methods might stimulate the further detailed investigation of spatial and functional relationships between structures immunopositive for selected neuroanatomical markers.