Re-evaluation of epoxy resin sections for light and electron microscopic immunostaining.

Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, alpha-catenin, and beta-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane.     

A rapid method for removal of detergents from protein solution

A simple and rapid technique is described for the removal of Triton X-100, deoxycholate, and cholate from protein solutions. The method involves a 2-min centrifugation of the sample on a Bio-Beads SM-2 bed prepared in a microcentrifuge tube and is suitable for multiple assays of 0.05- to 0.45-ml samples. Another advantage of this method is the high recovery of proteins without dilution of the sample.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

Improved staining procedures for semithin epoxy sections of plant tissues.

Improved and reliable methods are described for staining semithin sections of plant materials fixed in glutaraldehyde-osmium and embedded in epoxy resins. One-micron sections are fixed to slides, stained with a two-solution hematoxylin procedure or with a methylene blue-azure A combination, counterstained in aqueous safranin O, cleared, and mounted permanently. Basophilic tissue components are stained gray to black by the hematoxylin and blue or purple by the methylene blue-azure A combination; cell wall structures are colored by the safranin. With the procedures recommended, stains are sharp and intense, sections are flat, wrinkling and loss are held to a minimum, and unsightly precipitates do not form.     

A ruthenium red-toluidine blue procedure for staining epoxy sections in the light microscopy.

Ruthenium red (RR) has been widely used as a fixation additive for electron microscopy on the basis of its capacity to retain acid mucopolysaccharide residues of the cell surface coat. Little is known about the properties of this compound as a direct staining agent for epoxy-resin embedded material. In this study, semithin sections of Epon-infiltrated muscle tissue samples have been treated with 1% RR followed by counterstaining with 1% toluidine blue. This 2 step staining procedure has proven to be simple, rapid, and reliable and to give a dramatic improvement in image resolution and contrast. Thus, we believe that histochemical procedures employing RR may find in the future interesting applications for the direct staining of epoxy-resin embedded tissues.     

Preparation of serial sections of arthropods using 2,2-dimethoxypropane dehydration and epoxy resin embedding under vacuum

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used.