Affixing Plant Sections without Protein Based Adhesives for Protease Histochemistry

Abstract

To submit a section of plant tissue to histochemical analysis using protease, the protein based adhesives which keep the slices attached to the slides must be replaced because they are attacked by the enzyme and the slices are washed off the slides. We devised a method to keep the slices attached to the slides during histochemical extractions and subsequent staining. Slides are frosted on two lateral zones by spreading on them a fluoride paste composed of 15 g barium sulfate, 15 g ammonium difluoride, 8 g oxalic acid, 40 ml glycerine and 12 ml deionized water using a thin paint brush. After removing the paste with tap water and drying the slides, the sections are placed on the central clear zone of the slide and covered with an ethyl-cellulose film that keeps the slices in place and allows the reagents to act through it. To do this, the slides are dipped into 0.5% ethyl cellulose (ETC) prepared in a 4:1 mixture of toluene and absolute ethanol. The ETC coating is layered three times to improve its firmness and its ability to retain the slices on the slides. To obtain perfect adhesion, the slide should be oven dried (40-50 C for 10-15 min) to remove any trace of humidity before applying each layer of ETC. Subsequently the sections can be extracted and stained without undue loss of material.