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The ISH procedure applied on MNC in this study has previously been carefully evaluated and applied to detect mRNA in frozen tissue sections (194, 201-203). We found this method to have the necessary sensitivity to identify cytokine mRNA positive MNC among lo5 cells that were applied per slide per probe. The variation between duplicate applications was low (<lo%). Since the patients’ blood MNC usually were used for the study of multiple cytokines, frequently both without culture and after culture in presence of various antigens and without antigen, thereby leading to shortness of cells, most of the results presented in this study are based on single applications per probe. Because of the low numbers of CSF MNC usually available, only single applications per probe of CSF cells were evaluated. In most instances, MNC from a patient (MS/ ON and/or MG) and a control (OND or healthy subject) mere examined in parallel. The low numbers of cytokine mRNA expressing cells which were almost always found in the controls, strongly contrasting to the high numbers observed in many of the patients with neuroinflammatory diseasess, clearly indicate that the OND patients or healthy subjects represented acceptable internal controls. The specificity of the ISH was ascertained by the inclusion of a control probe with a similar length and guaninekytosine ratio. This control probe was always included on the study of cells from each patient. The results obtained with this probe were practically always negative. In those exceptional cases where positive cells were detected, their numbers were low (1-2 per lo5 MNC). Many factors may influence on the results of ISH, including pH, temperature, and concentration of the developing solution. We observed, however, that appropriate handling of the cells is crucial for the results. We found that ISH should be performed on a standardized number of cells, and applied 1 X lo5 MNC per slide and probe. With reference to the low numbers of CSF MNC available, cell numbers down to 5X103 were accepted, but the results obtained are less precise compared to those from examination of the standardized number of 1 X lo5 MNC. For evaluation of cytokine mRNA expression

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