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In situ hybridization methods for mouse whole mounts and tissue sections with and without additional β-galactosidase staining.

Methods in molecular biology (Clifton, N.J.) (2013-12-10)
Yoshihiro Komatsu, Satoshi Kishigami, Yuji Mishina
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.

MATERIALS
Product Number
Brand
Product Description

Roche
Anti-Digoxigenin-AP, Fab fragments, from sheep
Roche
Protector RNase Inhibitor
Roche
BM-Purple, Roche, pkg of 100 mL, solution
Roche
T3 RNA Polymerase, from Escherichia coli HB101
Roche
DIG RNA Labeling Mix, sufficient for 20 reactions, solution
Roche
T7 RNA Polymerase, from Escherichia coli BL 21/pAR 1219
Roche
Blocking Reagent, For nucleic acid hybridization and detection
Roche
SP6 RNA Polymerase, from Escherichia coli BL 21/pSR3