Riboprobes digoxigenin-labeled

The desired concentration is approx 2 pM digoxigenin-labeled riboprobe/ mL hybridization buffer (usually this works out to be 2-6 pL probe/50 pL buffer)... 

Tissue sections are obtained from rats that are killed by decapitation. The brains (or other required tissue) are rapidly removed and frozen in isopentane that has been precooled (-30°C) on dry ice. Sections (14 pM) are cryostat-cut and mounted (two sections per slide) under conditions that minimize RNase contamination. Mounting sections close to the bottom of the subbed slides will conserve reagents required for double-label ISH. Multiple adjacent series of sections are obtained, depending on need. For example, it is advisable to take a series for each [ Sj-labeled riboprobe both alone and in combination with each appropriate digoxigenin-labeled riboprobe. Slide-mounted sections are sorted at -20°C (in cryostat or cold room) prior to the assay to obtain the required number of near adjacent series. Slides brought to room temperature cannot be... 

Digoxigenm-labeled riboprobes are both transcribed (using the DIG RNA Labeling Kit [Boehrmger Mannheim]) and quantitated as described by the manufacturer (see Note 5). Store digoxigenin-labeled riboprobes at -70 C (see Note 6). 

It is very important to handle and store the digoxigenin-labeled riboprobes with care to avoid degradation by RNase contamination The probes should be stored at -80°C with an RNase inhibitor for long term use and can be stored at -20 C for daily use... 

More background information can be found in two books dedicated to ISH (11,12). Note that retinoid receptor or binding-protem transcript distribution may also be analyzed by whole-mount ISH of digoxigenm-labeled riboprobes, a method that is particularly informative at early stages of development and is described in Chapter 5 and in refs. 12 and 22, In Chapter 18, Xu and Lotan describe the use of digoxigenin-labeled RAR riboprobes for ISH of paraffin-embedded tissue sections. 

Partial alkaline hydrolysis reduces the nboprobe to smaller molecules, which are believed to reach target RNA more efficiently (7) Some researchers do not perform alkaline hydrolysis of the riboprobes. In particular, this step is omitted in the case of digoxigenin-labeled whole-mount ISH probes, at least up to 1 kb (12,22). We recommend systematically performing the alkaline hydrolysis of S-labeled probes. 

Slides are placed in hybridization trays containing Whatman filter paper soaked in standard saline citrate (SSC)/formamide solution. Riboprobe hybridization buffer is applied to tissue using a repeater pipet, and slides are cover slipped. The cocktail of digoxigenin and p S]-labeled ripobrobe hybridization buffer (or the [ Sj-labeled rihoprohe hybridization buffer alone) is appUed to tissue in fixed voiumes determined by the size of the cover slip required to cover the sections (i.e., 50 pL per 30-mm cover sUp for sections mounted one or two per slide or 100 oL per 5- mm cover sUp for sections mounted three or four per sUde). Slides are placed in a humidihed chamber and incubated overnight at 52-55°C. 

This chapter describes a nonradioactive method for the localization of mRNA in whole mouse embryos. It employs riboprobes labeled with digoxigenin, a steroid-like moiety not found in animal tissue. Digoxigenin-containing probe is visualized with a conjugate of antidigoxigenin Fab and alkaline phosphatase and colorimetric staining. The results are visualized in three dimensions, hence subtle patterns can be visualized without laborious sectioning. 

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