Tissue printing for peroxidases associated with lignification

Peroxidases with syringaldazine oxidase activity have been claimed to be specifically involved in lignification. The use of syringaldazine as a substrate presents problems of color diffusion and fading. We report a method for timing color development of syringaldazine peroxidase reactions in tissue prints, and conditions under which diffusion is minimized. The best time interval in which the tissue print and background gave significant contrast was determined using ELISA plates in which impressions of the tissue could be made, mimicking reactions on a membrane. This technique permitted simultaneous reading of a large number of samples and blanks, rendering enough data points for statistical analysis. Optimum development time for syringaldazine peroxidase reactions was between 10 and 20 min. Color diffusion and smearing of syringaldazine peroxidase reactions in tissue prints were minimized when prints were covered with a strip of membrane before adding the substrate mixture.