Sodium phosphate buffer

100 μl of overnight cultures grown in BHI broth, were fixed onto objective slides, Gram stained and processed for microscopic analysis. Slides were examined and photographed with a Leica DM4000B digital microscope through a 100x/1.3 oil-immersion objective. For electron microscopy, cells were harvested from overnight BHI cultures by centrifugation (6 000 g at 25°C for 5 min), fixed for 2 h at room temperature in 2.5% glutaraldehyde (Electron Microscopy Sciences) buffered in 0.1 M Sodium phosphate buffer (Sigma, Buchs, Switzerland), washed 3 times (0.1 M Sodium phosphate buffer), fixed, and stained with 1% osmium tetroxide (Sigma, Buchs, Switzerland). Samples were dehydrated in ascending concentrations of ethanol followed by dehydration with propylene oxide (Sigma, Buchs, Switzerland) and infiltration in 30% and 50% Epon (Epoxy embedding medium, Sigma, Buchs, Switzerland). From each cell pellet, 0.9 mm toluidine blue stained semi thin sections were produced. Representative areas were trimmed and subsequently 90 nm, lead citrate (Merck, Germany) and uranyl acetate (Serva Electrophoresis, Baden-Würtenberg, Germany) contrasted ultrathin sections were produced and viewed under a transmission electron microscope (TEM Phillips CM10) at the Institute for Veterinary Pathology, Zurich, (IVPZ) at the University of Zurich.

The activity of rat brain AChE was determined by the method of Ellman with a slight modification [31 (link)]. In brief, a mixture containing a 20 μl of sample solution, 200 μl of 0.1 mM sodium phosphate buffer (pH 8.0) (Sigma-Aldrich, USA), and 10 μl of 0.2 M DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)) (Sigma-Aldrich, USA) was mixed and incubated at room temperature for 5 minutes. Then, 10 μl of 15 mM acetylcholine thiochloride (ACTI) (Sigma-Aldrich, USA) was added and incubated for 3 minutes. The change in absorbance of light was measured at 412 nm for 3 minutes at regular intervals of 30 seconds using a microplate reader (iMark™ Microplate Absorbance Reader). The activity of AChE was calculated according to the equation below and expressed as nmol/min·mg protein.
$\begin{array}{c}\text{AChE\hspace{0.17em}activity}=\left(\frac{\mathrm{\Delta }A}{\left(1.36\times {10}^4\right)}\right)\times \left(\frac{1}{\left(20/230\right)}\right)C,\end{array}$ where ΔA = the difference of absorbance/minute and C = protein concentration of brain homogenate.

Syntaxin 10 siRNA or non-targeting control siRNA transfected monolayers were infected with C. trachomatis serovar L2 for 36 h. Infected monolayers were collected and fixed in 2% EM-grade paraformaldehyde plus 2.5% EM-grade glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM sodium phosphate buffer (Sigma Aldrich). Cells were processed for TEM as described previously (Beatty, 2006 (link)). TEM images were taken for two independent experiments. TEM images were used to quantify total numbers of organisms per inclusion, percentage of developmental forms in each inclusion and inclusion diameter. The diameter measurement was taken at the widest part of each inclusion. The results are displayed as μm units, based on the scale set from the electron microscope. All means and standard error of the means were calculated using GraphPad Prism 6 software.