H. Lee

Intrinsic forces alone are sufficient to cause closure of the neural tube in the chick

An isolated neural plate or a postnodal piece of early chick embryos, when cultured under appropriate experimental conditions, can undergo morphogenetic movements and form tubular structures closely resembling neural tubes of early chick embryos.

A method for preparing quick-frozen, freeze-substituted cells for transmission electron microscopy and immunocytochemistry

A quick‐freeze, freeze‐substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a ‘double‐fixation’ protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick‐frozen in Freon 22 and freeze‐substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon‐Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze‐substitution fixative. However, if glutaraldehyde is used alone as a freeze‐substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.

Diazepam inhibits the spreading of chick embryo fibroblasts

Diazepam (Valium/Roche), a benzodiazepine derivative, reversibly inhibited the spreading of chick embryo fibroblasts in a dose-related manner. This inhibition was accompanied by: 1 Fewer and less organized microfilament bundles. 2 A reduction in myosin fluorescence along these structures. 3 A reduction in the total myosin content of cells compared to corresponding controls. Overall results suggest that myosin plays an active role in the spreading of chick embryo fibroblasts.

Possible involvement of calmodulin in apical constriction of neuroepithelial cells and elevation of neural folds in the chick

Chlorpromazine and trifluoperazine HCl, antipsychotic drugs known to bind to calmodulin, reversibly inhibited elevation of neural folds by interfering with the contractile activity of apical microfilament bundles in developing chick neuroepithelial cells.

Studies on the mechanisms of neurulation in the chick: The intracellular distribution of Ca++1

Coated vesicles were found to accumulate Ca++ in neuroepithelial cells and may play a role in regulating the contractile activities of apical microfilament bundles during uplifting of neural folds in the chick.

A method for exposing the internal anatomy of small and delicate tissues for correlated SEM/TEM studies using polyethylene glycol embedding

A method for preparing and handling large, clean, distortion‐free cut surfaces through small and delicate tissues for correlated SEM/TEM examination is described. In this method, tissues are fixed according to conventional protocols; however, instead of critical‐point‐drying after fixation, tissues are first embedded in polyethylene glycol (PEG), a water‐soluble waxy solid. Tissue blocks are easily oriented and sectioned to the desired regions, immersed in a solvent to remove PEG, critical‐point‐dried, and examined with an SEM. The same tissue blocks can be reworked for TEM by immersing in propylene oxide and embedding in an epoxy resin.

A method for studying the three‐dimensional organization of cytoskeletal elements of cells: improvements in the polyethylene glycol technique

A method utilizing polyethylene glycol (PEG) as an extractable embedment for electron microscopy is described. Tissues are fixed according to conventional protocols, embedded in PEG, and sectioned. Sections (ranging from 100 to 500 nm in thickness) are mounted on grids, divested of their PEG matrix, critical‐point‐dried, and examined stereoscopically. This method greatly facilitates studies on the three‐dimensional organization of cytoskeletal and cytoplasmic contractile systems in both muscle and nonmuscle cells.

A new method for the preparation of double-fixed, quick-frozen, freeze-substituted cells for whole-cell transmission electron microscopy

A method is described in which quick‐frozen, freeze‐substituted, cultured cells can be prepared for whole‐cell transmission electron microscopy (WCTEM). This method is simple and reliable and can be carried out in most laboratories without special equipment. Cells grown on Formvar‐carbon‐coated nickel grids are quick‐frozen in Freon 22, freeze‐substituted in an ethanolic solution of glutaraldehyde, post‐fixed in osmium tetroxide and critical‐point‐dried. The quality of ultrastructural preservation using this ‘double fixation’ protocol is comparable to that of conventional WCTEM. However, the combination of quick‐freezing and WCTEM has the decided advantage over conventional WCTEM in that cellular activities are arrested almost instantaneously. Thus, this new method could potentially yield a more faithful representation of cytoarchitecture and is especially useful for studies on the structural basis of rapid cytoplasmic events which may remain undetected when using conventional fixation methods.