Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 1092, 1-15

In Situ Hybridization Methods for Mouse Whole Mounts and Tissue Sections With and Without Additional β-Galactosidase Staining

Affiliations

In Situ Hybridization Methods for Mouse Whole Mounts and Tissue Sections With and Without Additional β-Galactosidase Staining

Yoshihiro Komatsu et al. Methods Mol Biol.

Abstract

In situ hybridization is a powerful method for detecting endogenous mRNA sequences in morphologically preserved samples. We provide in situ hybridization methods, which are specifically optimized for mouse embryonic samples as whole mounts and section tissues. Additionally, β-Galactosidase (β-gal) is a popular reporter for detecting the expression of endogenous or exogenous genes. We reveal that 6-chloro-3-indoxyl-β-D-galactopyranoside (S-gal) is a more sensitive substrate for β-gal activity than 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). S-gal is advantageous where β-gal activity is limited including early stage mouse embryos. As a result of the increased sensitivity as well as the color compatibility of S-gal, we successfully combined β-gal staining using S-gal with in situ hybridization using DIG-labeled probes in both whole mounts and sections.

Figures

Fig. 1
Fig. 1
Comparison with X-gal and S-gal staining on time course dependency. S-gal and X-gal staining pattern at each time point. Upper panels are 30 min, middle panels are 2 h, and lower panels are overnight (O/N) color development. Two left panels are day 7.5 embryos each set has one embryo carrying ROSA26 (left) and one embryo as a negative control (right). The two right panels are sagittal sections of the mouse head for day 16.5 embryos carrying P0-Cre and ROSA26-reporter
Fig. 2
Fig. 2
Dual detection of β-gal activity and gene expression on whole mount and cryo-sectioned materials. (a) S-gal staining (magenta) and in situ hybridization signals (purple) were monitored for E11.5 mouse embryo. (b) S-gal staining and in situ hybridization signals were monitored by magenta and purple color in surrounding mesenchyme of incisor region at E14.5. Left panel is S-gal staining and right panel is double staining with S-gal and Msx1 in situ hybridization
Fig. 3
Fig. 3
Example for whole mount in situ hybridization at early stage of mouse embryonic development. Anterior and posterior marker gene expression analysis of mouse embryos at E7.5. Marker gene expression pattern of Shh, Cer1, Foxa2 (upper panels) and Brachyury, Wnt3a, Tbx6 (lower panels) respectively
Fig. 4
Fig. 4
Example for whole mount in situ hybridization at late stage of mouse embryonic development. Whole mount in situ hybridization was performed using Shh, Patch1, Patch2, and P21 at E13.5 and E14.5. Shh, Patch1, and Patch2 start to express in the prospective palatal rugae at E13.5 but there is no expression of P21 on the palatal rugae at E13.5 and E14.5. Patch1, Patch2, and P21 are strongly expressed in the edge of the palate at E13.5
Fig. 5
Fig. 5
Example for section in situ hybridization by paraffin section. Section in situ hybridization was performed using Shh specific RNA probe. Sagittal paraffin section of craniofacial region at E18.5. Note that Shh expression is specifically detected in hair follicle (upper panel) and pre-ameloblasts (lower panel)

Similar articles

See all similar articles

Cited by 12 PubMed Central articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback