Staining Paraffin Extracted, Alcohol Rinsed and Air Dried Plant Tissue with an Aqueous Mixture of Three Dyes

Abstract

A staining solution containing alcian blue 8GX, Bismarck brown Y and safranin O was prepared with 0.1 M sodium acetate buffer, pH 5.0. Paraffin was extracted with MicroClear® solvent from 10 μm tissue sections mounted on slides. Paraffin solvent was removed by rinsing with isopropanol, and tissues were air dried. Slides with bare dry tissue sections were immersed in the triple stain and structures could be distinguished within 30 min as follows: nonlignified cell walls, blue; lignified cell walls, nuclei and chloroplasts, red; and cuticle, brown or yellow-brown. Excess staining solution was removed by rinsing with tap water, and the tissues were air dried again. Coverslips were affixed with resin over the stained dry tissues. This novel procedure was tested with immature tomato fruit, mature apple fruit, and various leaf and stem specimens of dogwood, laurel, pawpaw, poinsettia and zonal geranium.