Bx60f microscope

All the solutions used for staining the slides and for image acquisition were prepared with a 0.2 μm filtered outlet of the wastewater treatment plant. This allowed to minimize changes in osmotic and ionic forces and to avoid the detachment of bacteria on the substratum.
Once carefully removed from the reactor, the slides were rinsed and stained in a DAPI (4’,6-diamidino-2-phenylindole) solution. Microscopic observations were done with an Olympus BX 60-F microscope. The acquisition was realized with a CCD Nikon Digital Camera DXM 1200F. Images with a dimension of 1280x1024 pixels², equivalent to 0.013 mm², were acquired in lossless RGB tif format. The acquisition parameters such as exposure time, sensitivity, use of UV filter (excitation filter: 330–385 nm, barrier filter: 420 nm) were kept constant for a given material and a given experiment. Sixty images equivalent to 0.78 mm² were recorded per slide.

A segment of the left lung was fixed in Histofix (Histolab) and embedded into paraffin and sectioned (3 µm) with a microtome. The tissue sections were placed on slides (Superfrost Plus; Fisher Scientific) and deparaffinized in serial baths of xylene and ethanol followed by staining using Mayer hematoxylin and 0.2% eosin (Histolab) or picrosirius red staining kit (Abcam, Cambridge, United Kingdom). The stained slides were imaged using an Olympus BX60F microscope with an SC50 camera.

The pancreas of WT and AIM2^−/− mice was excised and fixed for 24 h by means of 4% paraformaldehyde (PFA). In addition, isolated islets from WT and AIM2^−/− donor mice were incubated for 45 min at 37 °C in 100 µL HepatoQuick^®, 50 µL human citrate plasma, and 10 µL 10% CaCl₂ solution. The resulting clot was also fixed for 24 h in 4% PFA. The PFA-fixed specimens were embedded in paraffin and 3-μm-thick sections were cut.
Then, the sections were stained with antibodies against insulin (1:300), glucagon (1:300), somatostatin (1:300), CD31 (1:300), MPO (1:300), CD68 (1:300), and CD3 (1:300) and visualized by secondary antibodies. We used Hoechst 33342 and hematoxylin to stain cell nuclei. The sections were examined by means of a BX60F microscope (Olympus). The quantification was carried out by FIJI software (NIH) and is given in % of all islet cells.