COMBINATION OF ALKALINE-PHOSPHATASE IN-SITU HYBRIDIZATION WITH IMMUNOHISTOCHEMISTRY - COLOCALIZATION OF CALRETININ-MESSENGER-RNA WITH CALBINDIN AND TYROSINE-HYDROXYLASE IMMUNOREACTIVITY IN RAT SUBSTANTIA-NIGRANEURONS

We describe a method to combine non-radioactive in situ hybridization using alkaline phosphatase (AP) labelled oligonucleotide-probes with i mmunohistochemistry on the same thin paraffin section. The simultaneou s detection of calretinin-mRNA and calbindin- or tyrosine hydroxylase- like immunoreactivity in neurons of rat substantia nigra, pars compact a, was used as a test system to develop the method. Brains were fixed by perfusion with 4% paraformaldehyde and embedded in paraffin. Five-m u m-thick sections were processed for non-radioactive in situ hybridiz ation with a 33-base alkaline phosphatase conjugated synthetic oligonu cleotide complementary to calretinin mRNA. After hybridization and col our reaction to visualize calretinin mRNA, sections were incubated wit h antibodies against calbindin D-28K or tyrosine hydroxylase. Immunore action was visualized using the avidin-biotin-complex-technique and di aminobenzidine. As the colour of both reaction products differ markedl y, the distribution of calretinin mRNA-containing neurons (purple-blue , alkaline phosphatase product) and calbindin/tyrosine hydroxylase imm unopositive cells (brown peroxidase product) could be differentiated e asily on the same section. Calbindin- and tyrosine hydroxylase-like im munoreactivity was found in the majority of calretinin mRNA-containing cells within the substantia nigra, pars compacta, indicating that in this nucleus a proportion of the dopaminergic neurons contain both cal cium binding proteins calbindin and calretinin. In conclusion, non-rad ioactive in situ hybridization using alkaline phosphatase labelled oli gonucleotide probes can be readily combined with immunohistochemistry.