In-situ enzyme histochemistry on plastic-embedded plant material. The development of an artefact-free β-glucuronidase assay


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An in-situ enzyme histochemical method is described that preserves the tissue and cell structure as well as the enzyme activities. Flower buds at different developmental stages from wild-type and transgenic Brassica napus plants, the latter containing the GUS gene under the control of a tapetum-specific promoter, were used as starting material. The method is based on the following principles: processing of the tissue on crushed ice, no fixation but a pretreatment with spermidine, partial dehydration with acetone, and a final embedding in a water-miscible glycol methacrylate resin at 5°C. This method was used to set up a sensitive p-glucuronidase histochemical assay with a high resolution. A succinate dehydrogenase assay was included as a control for tissue and cell viability as well as a standard for the relative metabolic activities between different tissues and cell types.