In situ Hybridization of Barley Grains

Barley grains were fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline, pH 7.3, overnight at 4 °C. After dehydration by an ethanol series, samples were passed through a graded ethanol–methacrylate series and polymerized for at least 48 h in UV light (20 °C). Cross-sections (7 mm) were prepared and mounted on silane-coated slides (Sigma-Aldrich). Gene-specific fragments were amplified by PCR using gene-specific primers containing T3- and T7-promoter sequences (Table S6). Fragments were labelled with Digoxigenin (DIG) by in vitro transcription using T3- and T7-polymerase according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). After the purification of riboprobes, the efficiency of DIG labelling was verified by dot blotting and the probes (100 ng RNA) were denatured and treated with RNAse inhibitor for hybridization
Hybridization and immunological detection were performed after Drea and colleagues [133 (link)]. The hybridization signals were detected by an alkaline phosphatase-conjugated DIG antibody and visualized with 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Roche, Penzberg, Germany).