Transmission Electron Microscopy of Bee Tissues

Five honey bees were collected from each of the three colonies maintained in an incubator at 96 h after IAPV infection. The midgut and trachea were dissected and removed as mentioned above, and were prepared for transmission electron microscopy (TEM) as published previously by Pilgrim et al. [32 (link)]. In brief, tissues were dissected into 2% (wt/vol) paraformaldehyde containing 2.5% (wt/vol) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), which was as fixative. Then, heavy metal staining consisting of ddH₂O was performed using 2% (wt/vol) OsO4, followed by 1% (wt/vol) tannic acid and then 1% (wt/vol) aqueous uranyl acetate. To prevent precipitation, washing the tissue with ddH2O between each staining step was carried out. A Pelco BiowavePro (Ted Pella Inc., Redding, CA, USA) was used to perform fixation and staining steps at 100 W 20 Hg, for 3 min and 1 min, respectively. Dehydration was in a graded ethanol series before filtration and embedding in medium premix resin (TAAB, Reading, UK). For TEM, the tissues were cut into 70 to 74 nm serial sections and collected on Formvar-coated (0.25% (wt/vol) in chloroform; TAAB, Reading, UK) Gilder 200 mesh copper grids (GG017/C, TAAB, Reading, UK) using a UC6 ultra microtome (Leica Microsystems, Wetzlar, Germany). Images were acquired on a 120 kV HT7700 (HITACHI, Tokyo, Japan).