Tyramide Signal Amplification coupled with multiple immunolabeling and RNAscope in situ hybridization in formaldehyde-fixed paraffin-embedded human fetal brain.

Several strategies have been recently introduced to improve the practicality of multiple immunolabelling and RNA in situ hybridization methods. We present a modified hybrid protocol of recently described complex detection strategies: (1) elution of antibodies prior to second round of staining (2) use of integrated polymers of HRP with secondary antibodies, and (3) tyramide signal amplification of multiple immunofluorescence labelling, to achieve a high sensitivity sequential multiple labeling using antibodies from the same species. A modified protocol of the novel RNAscope in situ hybridization method, including coupling with immunofluorescence on sections of early human fetal brain, has also been developed. These two techniques, when properly optimized, were highly compatible with routine formaldehyde-fixed paraffin-embedded tissue that preserves the best morphological characteristics of delicate fetal brain samples, allowing high power signal amplification for detection of protein and mRNA of genes that are sparsely expressed in the human fetal telencephalon.