In Situ Hybridization of Maize Stamens

Stamens were sampled before anthesis and fixed in formaldehyde–acetic acid–ethanol, dehydrated in an ethanol series, cleared with xylene, and embedded in paraffin as described previously [24 (link), 25 (link)]. Paraffin-embedded tissues were cut into 10-μm-thick sections and placed on slides coated with poly-L-lysine (Sigma). Digoxigenin (DIG)-labeled antisense and sense RNA probes were transcribed from either XhoI- or XbaI-digested pEASY-ZmCPK32ISH using either T7 (antisense) or SP6 (sense) RNA polymerase (Roche), respectively. In situ hybridization was performed as described previously [25 (link)]. After the enzyme-catalyzed color reaction, an insoluble blue precipitate was observed. Slides were visualized with a Zeiss Axioscop 40 microscope and photographed with an Mrc5 camera (Zeiss).