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.
In situ hybridization Lipophilic dye Immunohistochemistry Neuronal tracing
This is a preview of subscription content, log in to check access
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Confocal images were obtained at the University of Iowa Carver Center for Imaging. We thank the Office of the Vice President for Research (OVPR), University of Iowa College of Liberal Arts and Sciences (CLAS), and the P30 core grant for support (DC 010362). This work was in part supported by a NASA base grant (Bernd Fritzsch) and 1R43GM108470-01 (Gray, Fritzsch).
Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98(2):81–85CrossRefPubMedGoogle Scholar
Koopman P (2001) In situ hybridization to mRNA: from black art to guiding light. Int J Dev Biol 45(3):619–622PubMedGoogle Scholar
Wallner GN, Amann R, Beisker W (1993) Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14(2):136–143CrossRefPubMedGoogle Scholar
Pan N et al (2012) A novel Atoh1 “self-terminating” mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability. PLoS One 7(1):e30358CrossRefPubMedCentralPubMedGoogle Scholar
Fritzsch B, Glover J (2006) Evolution of the deuterostome central nervous system: an intercalation of developmental patterning processes with cellular specification processes. Evol Nerv Syst 2:1–24Google Scholar
Duncan J et al (2011) Combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology. J Vis Exp 49:2451PubMedGoogle Scholar
Hadjieconomou D et al (2011) Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat Methods 8(3):260–266CrossRefPubMedGoogle Scholar
Hama H et al (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14(11):1481–1488CrossRefPubMedGoogle Scholar