Cultures grown and embedded in situ on Spurr discs, a low viscosity epoxy resin for electron microscopy

The ultrastructural study of cellular inter-relationships in vitro requires methods of embedding in situ. The present technic combines the advantages of growing the cells directly on the substrate with subsequent embedding in situ. In addition it uses Spurr medium which provides qualities of low viscosity, high penetrability and easy handling.     

Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

A reliable epoxy resin mixture and its application in routine biological transmission electron microscopy

Although there are many papers in the literature on materials and procedures for the embedding of tissue for transmission electron microscopy (TEM), most recent publications have emphasized techniques for specialized applications. Although these may in many cases also be suitable for routine applications in addition to the specialized applications for which they were developed, this may not be clear from the literature. This paper describes the formulation and suggested procedures for the use of an epoxy resin mixture which has been routinely used by novice and experienced workers with success for a wide variety of biological TEM investigations in a multi-user multi-disciplinary EM facility. Results are given of the use of this embedding medium in the investigation of a variety of tissue types.     

Direct embedding in epoxy resin of cells attached to cellophane

Macrophages were collected from mice or rats after subcutaneous implantation of cellophane or grown in Leighton tubes in which the flying coverslip was replaced by a strip of cellophane. The cellophane and attached cells were prepared for electron microscopy, embedded directly in epoxy resin and then sectioned. The cutting qualities of cellophane are adquate for ultrathin sectioning and permit the ultrastructural examination of the attached macrophage monolayer. The technique can be used for other cell types.     

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.