For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

Foreign antigens or mitogens initiate activation of T lymphocytes through antigen-specific receptors. After antigen-receptor interaction, T cells release a lymphotrophic hormone, interleukin 2 (IL2), and high-affinity IL2 receptors (IL2R) are progressively expressed on their surfaces. When evaluated under the scanning electron microscope after specific immunolabeling with colloidal gold markers, the density of expression of the 55 kd low-affinity IL2 receptor (Tac) increases in a time-dependent manner, reaching a maximum 72 hours after the onset of phytohemagglutinin (PHA) activation. In contrast, the number of cells expressing the 55 kd molecule reaches a maximum by 24 hours and remains constant through 72 hours. Immunogold labeling allows simultaneous determination of IL2 receptor density at the level of each individual cell and of the percentage of IL2 receptor-positive cells in any cell population under study. The rank order of receptor expression on the surface of established culture cell lines correlates with results reported from other laboratories with immunofluorescence and Scatchard analysis of binding of radiolabeled IL2. Although at high density of receptor expression immunogold labeling with 40 nm gold markers detects significantly fewer sites than radiolabeling, the method appears very sensitive in identifying low levels of expression. This results from 1) the extremely low non-specific background of the immunogold labeling technique and 2) the analysis of single cells rather than cell populations. Our results indicate that 2 cell lines, reported as negative for the expression of the 55 kd IL2 receptor (YT2C2 and MLA144), express low numbers of this molecule as demonstrated by immunogold labeling.

Effects of halothane on arrhythmias induced by myocardial ischaemiaThe effect of halothane on arrhythmias induced by ischaemia was investigated in rats, isolated perfused rat hearts, and pigs. Responses to the occlusion of the left anterior descending coronary artery were determined in groups (n = 9) of chronically prepared rats" treated with no halothane, 0. This laboratory has previously reported that halothane anaesthesia diminished ischaemia-induced arrhythmias when compared with pentobarbital anaesthesia. ~,2 Subsequently we showed that ischaemia-induced arrhythmias were also reduced in incidence, severity, and duration by halothane when compared with conscious rats or rats receiving fentanyl anaesthesia) This antiarrhythmic action of halothane was dose-dependent, and was much greater than other halogenated anaesthetics (methoxyflurane, enflurane, isoflurane, chloroform) thus demonstrating that halothane reduced severe arrhythmias independently of the anaesthetic state. 4 Other workers have demonstrated that halothane has antiarrhythmic actions against ischaemic arrhythmias in dogs. 5'6 However, the mechanism of halothane's antiarrhythmic action in rats and dogs remains unknown and the clinical significance of the findings uncertain 4,6,7To investigate the mechanism of action and the clinical relevance of the antiarrhythmic actions of halothane, we addressed the following questions.(1) Does halothane have antiarrhythmic actions when administered after coronary artery occlusion? If so, similar compounds should be investigated as a possible treatment for ischaemic arrhythmias, independent of their anaesthetic actions, and to provide clues into the mechanism of arrhythmogenesis. The time of onset of the antiarrhythmic action also helps to determine if the drug is acting in the perfused, non-perfused or border regions of the ischaemic zone.(2) Does halothane have antiarrhythmic actions in isolated perfused hearts? Answers to this question help define mechanisms of antiarrhythmic action, i.e., does it involve direct mechanisms, or indirect ones mediated by the CNS, endocrine or cardiovascular systems.(3) Is the antiarrhythmic action species dependent? By testing for antiarrhythmic actions in the pig, a species with a similar cardiovascular system to man, the experimental f'mdings can be better extrapolated to man. 23 Methods Study I Post-occlusion halothane in chronically prepared ratsUnder halothane anaesthesia rats were prepared with a left

Taking advantage of the high elemental contrast of particles of colloidal gold observed in the backscattered electron imaging(BEI) mode of the SEM (1,2), the human T lymphocyte was chosen as a model system to study the potential value of immunogold labeling for the quantification of cell surface expressed molecules. The CD3 antigen which is expressed on all human T lymphocytes and is readily identified by the LEU-4 murine monoclonal antibody (Becton Dickinson, Mountain View, CA) followed by a gold conjugated goat anti-mouse Ig polyclonal antibody was chosen as a model target antigen. When quantified by non-EM methods, using radio-iodinated probes or FACS analysis, approximately 30,000 to 50,000 copies of this antigen per cell are enumerated.The following observations were made while attempting to quantify the same molecule by SEM after specific immunogold labeling:Imaging in the SE vs BE mode: The numbers of gold markers counted in the secondary electron (SE) imaging mode are considerably lower than those counted on the same cells in the backscattered electron (BE) imaging mode.