Routinely used procedures for chemical fmtion often fail to preserve delicate membrane-bounded tubular structures in a variety of cell types. Fixation procedures commonly employed in immunocytochemical studies for localization of structural proteins, such as those found in cytoskeletal elements, may also degrade these tubular structures. Here we desaibe a procedure that p " s the elaborate tubular lysosome system found in stimulated macrophages and allows
Chromosome frequency distribution and cellular DNA estimations in different established mosquito cell lines were studied. These cell lines exhibited a wide range of cell types with a diploid stem-line comprising 50-55% and a haploid substem-line comprising 12-30% of the population. Estimation of cellular DNA contents by impulse cytoflowmetry and by Feulgen cytophotometry supported these observations. Because of their low diploid counts, these cell lines cannot be classified as diploid.
Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/− 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/− 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/− 6.27% for PL+F and 47.2% +/− 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/− 8.89% for PL+F and 39.6% +/− 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a ‘pre-defined’ apical location; (ii) translocated to a ‘newly defined’ basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.
24-48 h, and the solution was then replaced with 0.5 M EDTA, 0.5% paraformaldehyde, or 1% paraformaldehyde, each in CMF-PBS. Vials were stored at 4°C at all times. We can report that 11 months storage in 0.5 M ETDA, and 19 months in 0.5% and 1% paraformaldehyde, both soft-tissue and skeletal metastases still fluoresce (Figure 2, A and B). Under these conditions, a few metastases lost considerable fluorescence and were only slightly visible above background. By contrast, the majority of samples stored concurrently in 4% paraformaldehyde no longer fluoresced.Background auto-fluorescence commonly increases following fixation, and the intensity of GFP fluorescence is sometimes reduced compared to fresh tissue (Figure 2, C and D). Nonetheless, maintenance of fluorescence, along with relatively good preservation of cell morphology when tissues are routinely sectioned, renders this inconvenience acceptable for most uses (Figure 2, E-G). This technique now provides investigators adequate time to thoroughly examine tissues in largescale experiments involving several replicates in multiple experimental groups. The safety of 0.5 M EDTA for both decalcification and storage also eliminates the need for additional solution changes and enables immediate histological processing of archived samples, including use for standard histological staining.In conclusion, 0.5 M EDTA in CMF-PBS is capable of decalcifying murine bones in at least 24 h without harming GFP fluorescence and can be utilized for long-term archival of fluorescent specimens. Additionally, extended storage in 0.5%-1% paraformaldehyde maintains tissue fluorescence without concomitant decalcification.
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