Cm120 biotwin electron microscope

Vectors were prepared as described above. Samples were concentrated with 30000 molecular weight cut off (MWCO) spin columns and dried onto a gold grid and stained with 1% aqueous uranyl acetate. Images were obtained using a CM120 BioTwin electron microscope (Philips, USA).

For TEM analysis, samples were fixed in Karnovsky’s fixative overnight at room temperature followed by post-fixation in OsO4 for 1 h. Preparations were dehydrated in graded alcohols and embedded in low viscosity resin (TAAB Laboratory and Microscopy, UK). Ultrathin sections were mounted on Pioloform/formvar-coated slot grids, stained in uranyl acetate and lead citrate, and examined in a Phillips CM120 BioTWIN electron microscope.

Disrupted Malpighian tubules were smeared on microscope slides, air-dried, fixed in methanol (5 min), and stained with Giemsa solution (Accustain, Sigma-Aldrich; 45 min in a 1:10 dilution). Dried smears were embedded in Entellan (Merck). For scanning electron microscopy, cover glasses with smears of infected Malpighian tubules were air-dried and coated with gold particles using a Baltec SCD 040 sputter device. Micrographs were taken with a Quanta 200 electron microscope (FEI Company). For transmission electron microscopy, samples were fixed for several days with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C, rinsed with cacodylate buffer, and post-fixed for 1.5 h with reduced OsO₄ at room temperature without darkening (fresh 1:1 mixture of 2% OsO₄ and 3% K₄[Fe(CN)₆]). The samples were then rinsed with distilled water, and after dehydration in a series of ethanol (15 min each in 30%, 50%, 70%, 90%, 100%, 3 × 100% water free), they were embedded in Spurr’s resin^{55 (link)}. Ultrathin sections were stained with saturated aqueous uranyl acetate for 30 min in the dark, followed by Reynolds’ lead citrate^{56 (link)} for 5 min. During the latter, sodium hydroxide pellets were put next to the grids in order to remove CO₂ from the atmosphere and thus prevent precipitation of lead carbonate. The sections were investigated using a Philips CM 120 BioTwin electron microscope.

For TEM analysis, samples were fixed in Karnovsky’s fixative overnight at room temperature followed by post-fixation in OsO₄ for 1 h. Preparations were dehydrated in graded alcohols and embedded in low viscosity resin (TAAB Laboratory and Microscopy, United Kingdom). Ultrathin sections were mounted on Pioloform/formvar coated slot grids, stained in uranyl acetate and lead citrate and examined in a Phillips CM120 BioTWIN electron microscope.

For TEM analysis, tissues were fixed in Karnovsky's fixative overnight at room temperature followed by post-fixation in OsO₄ for 1 h. Preparations were dehydrated in graded alcohols and embedded in low viscosity resin (TAAB Laboratory and Microscopy, United Kingdom). Ultrathin sections were mounted on Pioloform/formvar coated slot grids, stained in uranyl acetate and lead citrate and examined in a Phillips CM120 BioTWIN electron microscope.

Cover glasses removed from positive squash preparations were used for scanning electron microscopy (SEM). They were air dried in order to prevent parasite loss during further preparation steps. The dried cover glasses were mounted on aluminium stubs with double-sided adhesive tape, sputter-coated with gold in a Balzers SCD 40 and observed using a FEI Quanta 200 ESEM.
For transmission electron microscopy (TEM), infected tubules were fixed in 2.5% glutardialdehyde in 0.1 M cacodylate buffer (pH 7.2) and stored in a fridge for several days. The fixed tissue was then washed three times in buffer, post-fixed in 1% osmium tetroxide (OsO₄) plus 1.5% potassium ferrocyanide (K₃[Fe(CN)₆]) for 1.5 h at room temperature, washed three times, dehydrated in a gradient of ethanol and embedded in Spurr’s resin (Spurr 1969 (link)). Ultrathin sections were contrasted with saturated aqueous uranyl acetate ([UO₂(CH₃COO)₂ · 2 H₂O]) for 30 min followed by lead citrate (C₁₂H₁₀O₁₄Pb₃) (Reynolds 1963 (link)) for 5 min. The sections were examined with a Philips CM 120 BioTwin electron microscope.