Combining Whole-Mount In Situ Hybridization with Neuronal Tracing and Immunohistochemistry

Despite the presence of several markers to study the expression of genes (eGFP, LacZ), in situ hybridization remains one of the most powerful techniques to analyze gene expression. While this allows cellular identification of the expression of a single gene and, using fluorescent in situ hybridization two or occasionally more genes, it is often necessary to combine this technology with assays of neuronal projection/morphology, protein expression using antibody staining, and histology for cytological details. Since each task has certain levels of false negatives, combining them in a single preparation can compromise further correlative studies due to loss of fluorescence, loss of antigenic epitope, or loss of tissue morphology. We have designed a protocol that, when performed in sequence, will enable the researcher to combine several of these technologies in the same sample saving time and sparing expense. By combining neuronal tracing, whole-mount in situ hybridization, immunohistochemistry, and histology one can extract a maximal amount of data with limited loss in fidelity of each technique and optimal data superposition for a more complete understanding of phenotypes.

Source: Springer protocols feed by Neuroscience - February 20, 2015 Category: Neuroscience Source Type: news