Zen 3.1 blue edition software

To investigate the viability of the cells in the cultured metatarsi, a Live/Dead assay (Abcam, Cambridge, UK) was carried out following the manufacturer’s instructions. Accordingly, metatarsi collected immediately after dissection and the establishment of cultures (day 0) and after 14 days of maintenance under standard culture conditions, were stained with 5× Live and Dead Cell stain in DMSO in 300 µL of 1× sterile PBS, and incubated for 10 min at RT. After the incubation, samples were imaged on a Leica DM4000 upright fluorescence microscope (Leica Biosystems, Nussloch, Germany) with a Zeiss mRM camera using Zeiss ZEN 3.1 blue edition software (Carl Zeiss Microscopy GmbH, Munich, Germany) with L5 (green fluorescence) and TX (red fluorescence) channels.
To quantify the levels of fluorescence indicative of live/dead cell labelling under these various conditions, images were opened on Zeiss ZEN 3.1 blue edition software (Carl Zeiss Microscopy GmbH, Munich, Germany) and the metatarsal area was manually traced. After tracing the area, the average fluorescence was obtained automatically by the software for both the FITC (live cells) and Texas Red (dead cells) channels. Graphs were then displayed by average fluorescence per µm² to allow for direct comparison between metatarsi with different lengths.

A qualitative method was used to categorise and tabulate the interaction of thioesters and esters with human (Figure 2) and mouse (Figure 3) brain tissue ChEs as shown using the KR histochemical method. For thioesters, the staining intensity was categorised as: o (no staining), + (weak), ++ (moderate), or +++ (strong). For esters, the reduction in AChE and BChE staining intensity was categorised as: - (no reduction), x (slight), xx (moderate), or xxx (strong). All tissue sections were analysed independently by two observers using brightfield microscopy on an Olympus BX50 microscope. Any discrepancies were jointly discussed until a consensus was reached. Stained tissue sections were photographed with a Zeiss Axio Scan.Z1 slide scanner with Zen 3.1 Blue Edition software (Carl Zeiss Canada Ltd, Toronto, Ontario, Canada). The photomicrographs were assembled into figures using Adobe Photoshop (CS 5, Version 12.0, San Diego, California, United States). The brightness of the photographs was adjusted to match the background of each image.

Biofilms were grown in μ-Slide slides (Ibidi, Gräfelfing, Germany) inoculated with ∼1 × 10⁵ cells in 500 μL of fresh BHI medium and incubated for 48 h at 37°C. The culture medium was changed after 24 h of biofilm growth. Biofilms were stained using the Live/Dead BacLight bacterial viability kit (Life Technologies, New York, NY, USA) and/or TOTO-1 iodide staining (Thermo Fisher Scientific, catalog [cat.] no. T3600; dilution, 1:1,000) for detection of free eDNA surrounding living and dead cells (85 (link), 86 (link)) and examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Data were analyzed with the ZEN 3.1 (blue edition) software (Zeiss).