Method

bodies (Cat. No. 11093272001, Roche Applied Science)

• RNase-free microfuge tubes (Eppendorf)

• Hybond N+ membranes (Amersham)

• DIG RNA Labelling Kit (SP6/T7) (Cat. No. 11175025001, Roche Applied Science)

1 Linearize the cloned plasmid with an appropriate restriction enzyme to give a synthesized transcript of 500-600 bp, an optimal length for DIG-labelled RNA probes.

2 Prepare DIG-labelled probes according to the manufacturer's instructions using DIG-UTP. Typically, 1 mg template DNA provides *10 mg DIG-labelled probe. The labelling efficiency can be determined by dot blot, using anti-DIG-alkaline phos-phatase and NBT/BCIP colour development substrate, according to the manufacturer's protocol. Labelled probes are stable for 1 year when stored at -20 °C. Avoid repeated freezing and thawing of probes.

5.3.1 Probe preparation

Probes for ISH can be either RNA or DNA (single- or double-stranded). RNA probes are particularly effective, and are generated from plasmids containing SP6, T7, or T3 RNA polymerase recognition sequences. Ideally, the plasmid should contain two such recognition sites, placed at either end of the DNA insert, so that

Pleiotrophin

Whole Mount Sections

Myosin Heavy Chain

Figure 5 Examples of ISH performed on EBs generated from the R1 line of mES cells, for the localization of muscle-associated transcripts. (A) Whole-mount ISH with DIG-labelled RNA probes generated against pleiotrophin. (B) Whole-mount ISH, and ISH on sections of EBs, with probes against myosin heavy chain (MHC). Dark staining indicates the location of pleiotrophin or MHC transcripts in these preparations. Probe orientations are indicated by ' +' for the antisense, and ' - 'for the sense probe.

both the identical (negative control) and complementary (experimental) probes to the target species can be synthesized by RNA polymerase. An important consideration is that hybrid formation in solution is proportional to the single-stranded probe's length; this however must be counterbalanced, in the context of cellular aggregates, with the relative ease of diffusion of smaller probes into the dense matrix surrounding the cell.

5.3.2 Whole-mount in situ hybridization of EBs (Figure 5)

ISH is a cornerstone of molecular analysis in developmental biology, having proven fundamental to the delineation of patterns of gene expression during development. It also represents the method of choice for visualizing mRNA molecules in circumstances where they are distributed in a non-random, topographical manner, as in EBs.

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