Triple immunofluorescence labelling of parvalbumin, calbindin-D-28k and calretinin in rat and monkey brain

This study presents novel techniques for the concomitant cytochemical detection of the calcium-binding proteins parvalbumin, calbindin-D-28k, 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-D-28k - 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-D-28k, 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.