Capsule-supporting ring: a new device for resin embedding of glass-mounted specimens.

The goal of specimen preparation for transmission electron microscopy is to obtain high-quality ultra-thin sections with which we can correlate cellular structure to physiological function. In this study, we newly developed a capsule-supporting ring that can be useful for resin embedding of glass-mounted specimens. The present device allowed us to re-embed a semi-thin section on a microscope slide into a resin block not only for efficient ultra-thin sectioning but also for a correlative light and electron microscopy. Similar to epoxy resins for morphological observations, semi-thin sections of low-viscosity hydrophilic resins, such as Lowicryl series, can be re-embedded into the resin, which can be useful for cytochemical gold labelling. A further application of the present device improved flat embedding of cultured cells on glass cover slips for electron microscopy, preserving in situ sub-cellular structures close to their native state. We practically describe the use of capsule-supporting ring and demonstrate representative micrographs as results.

Embedding thin plant specimens for oriented sectioning.

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.

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.

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.

Oblique sectional planes of block plastinates eased by Sac Plastination.

To find an oblique cutting plane of a plastinate, e.g. to cut gamma-nails in the femur, the Block Plastination technique was modified. After CT and MRI examination, the specimens were plastinated with the standard resin mixture E6/E12/E600. Instead of using a box to form a block during the casting and curing stage, we embedded the specimen in a sac made of polyester foil. A polymerized wooden block was attached to the specimen. The sac was wrapped with tape to the embedded specimen with the block. This approach limited the amount of required resin to the inner volume of the plastinate. Then, the plastination sac was put in the incubator for further polymerization and curing. When the foil was removed from the plastinated specimen, the wooden block served as a socket for the grip when sawing. The outer shape of the specimen remained visible. Doing so, the adequate cutting plane could be determined easily.

Design of lifting gear for a plastination laboratory.

Conventional lifting gear based on steel components can be safely used in a laboratory where acetone is used provided that: (a) the circumstances under which significant amounts of acetone vapour will be present are known and can be controlled and (b) operating practices are imposed to prevent the use of the lifting gear at times when acetone vapour may be present in explosive concentrations.

[Simplification of the production of plastination histologic preparations through the use of a grinding machine]. / Vereinfachung der Herstellung plastinationshistologischer Präparate durch Einsatz einer Schleifmaschine.

A new histological method basing on the plastination technique (v. Hagens, Tiedemann, Kriz 1987) has recently been developed and applied to research in human fetal development. This plastination histology shows some evident advantages compared with the classical histological techniques. As the application of this new method has been very time consuming up to now, only some special questions of basic research have been investigated. We here present the use of a grinding-machine, which can be used to partially mechanize the procedure of plastination histology.

Efficient embedding technique for preparing small specimens for stereological volume estimation: zebrafish larvae.

Stereological sampling regimes, in particular volume and number estimation, often require systematic uniformly random sections throughout a specimen. A method has been developed to increase the efficiency of preparing fish larvae for sectioning prior to histological or stereological analysis. Embedding a group of larvae in a resin block using this technique greatly reduces the quantity of sections produced and allows easy assessment of sample groups. Saving time in this way therefore makes stereology a more viable research tool.

Recognition of basic fuchsin prestained microfissures of intravital origin with fluorescence microscopy: validation of a shortcut.

For 70 years it has been suspected that not all microfissures in histological bone sections are artifacts, but that some are provoked in vivo through repetitive stress. The development of undecalcified bone techniques and of the bulk staining technique has established a method for demonstrating the existence of intravital cracks and enhanced the discrimination towards artifactual microfissures in the load-bearing skeleton. Recently the presence of intravital microfissures has also been ascertained in temporal bones by these techniques. Due to the fluorescent properties of basic fuchsin it is possible to use epifluorescence microscopy for analysis of microfissures after bulk staining with basic fuchsin. This provides a more steady microscopic background and a sharper delineation of surface level structures since no projection from lower levels interfere. Artifactual cracks, which in transmitted light microscopy may look like darkly stained intravital microfissures due to refraction phenomena, become invisible or colorless. Epifluorescence microscopy enhances the detection of both smaller and larger prestained intravital microfissures, and leaves only a minor part of the cracks without certain categorization. The epifluorescence mode of analysis has the further advantage of being independent of slice thickness, making feasible whole-specimen analysis by serial stepwise grinding. The present study shows that the number and the length of microfissures in the human otic capsule, counted and measured under the epifluorescence microscope, equals numerically the findings in light microscopy, enabling the routine use of this mode of analysis. This may prove to be of particular value in the research into the etiology and pathogenesis of otosclerosis as well as perilymphatic fistulae.