Nis br software

Cells were cultured for 24–48 h on glass coverslips, then were then washed twice in PBS, before being fixed with 3.7% ice-cold paraformaldehyde in PBS. Cell permeabilization was obtained using a solution containing 50 mM NH₄Cl, 1% BSA and 0.02% saponin. Saturation of epitopes was fulfilled by incubating 2 h with a solution containing 3% BSA and 3% Normal Goat Serum (NGS). Epifluorescence microscopy was performed with a Nikon TI-S microscope. Images were analysed using both NIS-BR software (Nikon, France) and ImageJ^© software 1.38I (http://rsbweb.nih.gov/ij). Fluorescent probes and conjugated antibodies were purchased from Fisher Scientific (France).

At post mortem, the full carcass of every mouse was fixed by immersion in 10% buffered formalin at room temperature (RT), after opening the thoracic and abdominal cavities for exposure of internal organs for over a week. A post mortem examination was carried out with the fixed carcass following a standard protocol and samples from the following organs were removed: brain, heart, liver, spleen, kidney, large intestine, thymus, pancreas and a skin sample from the inoculation area. Immediately following dissection, samples were further fixed in 10% formalin and embedded into paraffin wax. Additional samples were preserved in 10% formalin for immunofluorescence.
Sectioning and haematoxylin and eosin (HE) staining were performed according to standard laboratory procedures at the University of Surrey Veterinary Pathology Centre using an automated stainer (Sakura Tissue-Tek DRS 2000). Briefly, 4 μm sections were moved to distilled water before nuclei were stained with haematoxylin. Acid-alcohol (0.3%) was then applied, before rinsing in running water. The eosin stain was applied for 2 min before dehydration and mounting. Stained organ sections were examined under the light microscope and histological lesions were recorded. Images were captured directly from the microscope (Nikon NI series) using Nikon NIS-BR software.

Transfected neurons were fixed at DIV8 with 4% formaldehyde and 4% sucrose in phosphate-buffered saline (PBS) at room temperature for 10 min. Cells were then washed three times in PBS-CM (PBS, 1 mM MgCl₂, 0.1 mM CaCl₂), permeabilized with 0.2% Triton X-100 for 15 min, and washed one time with PBS-CM, before incubation with 0.2% gelatin for 30 min at 37°C. Next, neurons were incubated with primary antibodies diluted in 0.2% gelatin for 30 min at 37°C, and washed three times in PBS-CM. This was followed by incubation with secondary antibody diluted in 0.2% gelatin for 30 min at 37°C, and washing three times in PBS-CM. Finally, coverslips were mounted in Fluoromount (Invitrogen).
Fixed cells were imaged on: (1) a Nikon Eclipse 80i upright widefield fluorescence microscope, equipped with a Photometrics CoolSNAP HQ2 CCD camera and Nikon NIS Br software, using a Plan Fluor 40× N.A/1.30 oil objective; or (2) a Carl Zeiss LSM 700 confocal laser scanning microscope running ZEN2011 software, using a Plan-Apochromat 40×/1.30 oil DIC objective.