A quick preparative method for electron microscopy observations of delicate objects using alginate embedding medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

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 method for in situ embedding of cultured cells grown on plastic surfaces

Summary A method is described for the electron microscopic visualization of contact relationships between monolayer cells and the supporting surface of the plastic culture vessel.     

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.     

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.     

Better epoxy resin embedding for electron microscopy at low relative humidity.

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used; impregnate tissues using the following solutions 1) 70% alcohol - 5 minutes, 2) 95% alcohol - 2 x 15 minutes, 3) absolute alcohol - 3 x 20 minutes, 4) acetone - 2 x 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 812, 39.4 g; DMP-30, 2.6 g) - 1 hour, 6) acetone-epoxy resin 1:3 - 1 hour, 7) epoxy resin - 1 hour; complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.