Permissiveness of Guinea Pig Alveolar Macrophage Subpopulations to Acute Respiratory Syncytial Virus Infection In Vitro: Immunogold Electron Microscopy

In: Respiratory

24 Jul 2014

After exposure to RSV, as described above, AMs were fixed in 2.5% glutaraldehyde-containing sodium cacodylate buffer (0.1 M, pH 7.4) for 1 h at 4°C, postfixed in osmium tetroxide for 1 h at room temperature, and embedded in epoxy resin following standard methods. Ultrathin sections were cut with an ultramicrotome (Reichert; Vienna, Austria) and collected onto Formvar vinyl alcohol-vinyl acetate formal copolymer-coated nickel grids (Ted Pella, Inc; Redding, CA). Following treatment with sodium metaperiodate to unmask antigenic sites and blocking nonspecific binding by incubation with normal rabbit serum (5% in PBS, 20 min), the sections were incubated with a cocktail of mouse anti-RSV monoclonal antibodies optimally diluted 1:100 as recommended by the manufacturer (NCL-RSV2; Novacastra Laboratories; Newcastle Upon Tyne, UK). The sections were washed again with PBS and then incubated at room temperature for 30 min with a rabbit antimouse antibody (1:20) conjugated to 10-nm colloidal gold particles (Dako). Canadian pharmacy add comment After three washes in PBS, followed by a final wash in distilled water, the sections were contrasted with uranyl acetate and lead citrate. For control sections, incubation with the primary antibody was omitted. Sections were examined under a transmission electron microscope (Philips 400; N. V. Philips’ Gloeilampen-fabrieken; Eindhoven, The Netherlands).
Statistical Analysis
The data are presented as means ± SD of values obtained from five guinea pigs. Data were analyzed using a one-way analysis of variance, and a sequential rejective Bonferroni procedure was used to correct for multiple comparisons. A p value < 0.05 was considered to be statistically significant.
Results
Viability of AM Subpopulations After In Vitro Exposure to RSV
Hypodense and intermediate-density AMs showed no apparent loss in cell adhesiveness to the culture plates at 24 h postinfection with RSV. Some detachment (< 10% of adherent cells) occurred at this time point in RSV-exposed high-density AMs and in RSV-exposed blood monocytes. However, cell viability remained > 95% in all cultures at 24 h postinfection.


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