An economical and practical method for whole-mount in situ hybridization for mouse embryos and organs

Abstract

Whole-mount in situ hybridization (WISH) is a useful method for detecting specific gene expression patterns at their site of action during embryonic development. Traditional WISH methods are costly and suitable only for mouse embryos younger than 11.5 days. We present here an economical and practical in situ hybridization method using DIG-labeled RNA probes. We changed the conditions in several steps to make the WISH method suitable for whole mouse embryos from embryonic days 9.5 to 12.5 and for older stage mouse embryonic organs. We performed all steps in one microcentrifuge tube up to the staining steps to avoid losing or damaging the mouse embryos. We re-used the solutions and materials to make the method more economical and suitable for less sophisticated laboratories. We also performed β-galactosidase staining on Tb × 18 Cre/Rosa26/LacZ mouse embryos; the results agreed with the in situ hybridization results. Finally, we sectioned the specimens after hybridization and β-galactosidase staining; the results agreed with the literature.

Single-cell and spatial heterogeneity landscapes of mature epicardial cells

“…Whole-mount X-gal staining was performed as previously described [ 31 ]. In brief, 4% ( V / V ) paraformaldehyde (PFA) was used to fix samples for 15–60 min.…”

“…To visualize Tbx1 8 messenger RNA (mRNA), we performed whole-mount RNA in situ hybridization on mouse embryos as previously described through an optimized method [ 31 ]. The probe plasmids of Tbx18 were constructed by Kispert and co-workers [ 32 ].…”