Samples were viewed with an Axioscop 2 plus fluorescent microscope (Zeiss), images were
captured with a high resolution microscopy camera AxioCam HRc and AxioVision software. Germaria from ovaries of 10 flies were counted in each of the 4 groups. The total number of germaria analysed was about 850. The data were compared using a Chi-square test (χ2). Electron microscopy Fixation of the D. melanogaster ovaries was carried out using the method described previously [49, 35]. Briefly, 5 day-old females were dissected in 0.1 M phosphate buffer, pH 7.4, fixed in 2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer, pH 7.4, for 2.5 h. This was followed by washings in the same buffer and postfixation in 1% OsO4 and 0.8% potassium ferrocyanide for 1 h. After washings, samples were placed in 1% aqueous solution of uranyl acetate (Serva) for 12 h at 4 °C. buy Combretastatin A4 Then they were dehydrated in ethanol series and acetone, finally samples were embedded in Agar 100 Resin (Agar Scientific Ltd.). Ultra-thin sections were stained with
uranyl acetate and Reynolds lead citrate. They were examined with a transmission electron microscope (JEM 100 SX, JEOL). The number of flies analysed in each of the 4 groups was 8-12. Acknowledgements We thank Prof. S. O’Neill (The University of Queensland, Australia) for kindly supplying us with D. melanogaster stock. We are also grateful to the staff of the IC&G SB RAS, particularly to Dr. A.A. Ogienko for sharing her experience with AO-staining of the D. melanogaster ovaries, Prof. I.K. Zakharov for providing conditions for fly maintenance, MK0683 chemical structure A.N. Fadeeva for translating the manuscript from Russian into English. This work was supported
by the Program of Basic Research of the RAS Presidium “Biodiversity” (26.30), “see more Molecular and Cellular biology” (6.12) and a grant from the Russian Foundation for Basic Research. This article has been published as part of BMC Microbiology Volume 11 Supplement 1, 2012: Arthropod symbioses: from fundamental studies to pest and disease mangement. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2180/12?issue=S1. Electronic supplementary material Additional file 1: TUNEL in the germaria from ovaries of D. melanogaster. Three PAK5 groups of germaria are distinguished. A, B, the TUNEL-negative germaria from the ovaries of D. melanogaster w1118T and Canton ST, respectively. C, D, the TUNEL-positive germaria with 1-2 distinct puncta in region 2a/2b of the germarium from the same fly stocks, as in A, B. E, F, the TUNEL-positive germaria with clusters of bright spots. Region 2a/2b of the germarium is indicated by red brackets. Scale bars: 20 μm. (TIF 537 KB) Additional file 2: Cystocytes in region 2a/2b of the germarium from the wMel-infected D. melanogaster Canton S.