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A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 p g / d to 5 pg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed. o 1992 Wiey-Liss, Inc.Keyterms: SV40, large T antigen, indirect immunofluorescence, propidium iodide, multiparameter analysis Several reports (see references in 9, 10, and 15) have supported the view that an in-depth understanding of the mechanism of action of cell proliferation-regulating genes could be facilitated by flow cytometric measurement of their products as a function of the cell cycle. This can be achieved by dual-parametric analysis of gene expression and DNA content through simultaneous immunofluorescence and propidium staining. The fixation and staining techniques have worked well for quantification of abundant or moderately abundant intracellular antigens (9,lO). However, current techniques have limited sensitivity that makes analysis of low-abundance antigens difficult (e.g., ref. 13).Various cytometric studies have described techniques that can sensitively measure cell-surface antigens (copy number of a few hundred molecules) by indirect immunofluorescent labeling (7,27). Intracellular antigens have been less amenable to sensitive analysis (5,6,11,17). Besides background fluorescence, the limiting factors for antigen detection are incomplete permeabilization, non-specific binding of antibody, and propidium emission below 540 nm.One way to increase the sensitivity of a labeling assay is through further signal amplification by introducing additional steps of multilayered immunolabeling (19). The high affinity of biotin for streptavidin (Kd -M) has been utilized to amplify immunohistochemical staining using biotinylated secondary antibody and labeled streptavidin (16), in enzyme-linked immunosorbent assays (ELISA) (25) and for membrane receptor assays (12,22,26,27). Fluorochrome-labeled streptavidin has been used for flow cytometric detection of cell-surface antigens (2,12,26), RNA (3), and DNA polymerase ci (23).With the goal of gaining an insight into mechanisms of normal and abnormal growth regu...

Local anesthesia techniques are commonly used to perform the cataract surgery. Among these techniques, peribulbar anesthesia (PBA), when compared to retrobulbar anesthesia, is more effective and safe. However, PBA is not without risk. Complications may arise which if not recognized early and resuscitated, can be fatal. A 70-year-old Indian female was admitted for cataract extraction and lens implant in her left eye. Lignocaine sensitivity was done and showed no abnormal reaction. She developed generalized tonicclonic convulsions following peribulbar block. This case report attempts to highlight the need for a trained personnel, compliance with safety standards in anesthesia, awareness, and early recognition of the possible complications and their management.

Frontal-plane deformity of the proximal tibia in children has a variety of etiologies. There are also a number of described surgical techniques for correction of these deformities. The authors reviewed their early experience with the use of the Orthofix T-Garches external fixator for correction of 16 proximal tibial deformities in 14 patients. Mean age at surgery was 14.2 years. The most common diagnosis was adolescent Blount's disease. Average deformity was 12 degrees. Total treatment time averaged 13 weeks for those requiring lengthening and 10.8 weeks for those without. Complications included one ring sequestrum treated by curettage, and two patients with subsequent development of femoral deformity. Tibial anatomic axis averaged 1 degree of varus. This device can achieve excellent correction of deformity isolated to the frontal plane with few complications. It allows functional weight bearing and use of adjacent joints during treatment.