Xarosa digital camera

For each animal treated with [³H]-thymidine, a minimum of 45 semithin section were examined for the presence of [³H]-thymidine labeled cells and photographed under a Nikon microscope (Eclipse E800, Nikon with digital camera Nikon DS-Ri1). A cell was considered labeled if it had eight or more silver grains over the nucleus. The semithin sections containing the selected cells were re-embedded, and ultrathin sections were cut and examined under a FEI Tecnai G² Spirit transmission electron microscope (FEI Company, Tokyo, Japan). Images were acquired using Radius software (Version 2.1) with a XAROSA digital camera (EMSIS GmbH, Münster, Germany).

GSDMB constructs were visualized by immunofluorescence in transient transfected (72 h) and 4% paraformaldehyde-fixed cells with the indicated primary antibodies (Supplementary Table 2) as described before [42 (link)]. Confocal microscopy images were captured by LSM710 microscope (Zeiss) and processed by Fiji software (Image J 1.52). Alternatively, live cell tracking imaging was performed with doxycycline inducible vectors expressing GFP-tagged GSDMB constructs (Supplementary information). To evaluate mitochondrial morphology correlative light and electron microscopy (CLEM) procedures were performed in 23132/87 cells. Cells were seeded in a permanox Lab-Tek chamber slide (Nalge Nunc International) and transfected with doxycycline inducible vectors. After 6 h of transfection, cells were induced with Doxycycline at 200 ng/ml, incubated with red MitoTracker^TM Deep Red FM (ThermoFisher) for 30 min at 37 °C and fixed in 3% glutaraldehyde. The confocal images were acquired with a Leica TCS SP8 HyVolution II (Leica Microsystems). After fluorescence capture, slides were processed for transmission electron microscopy analysis with FEI Tecnai Spirit BioTwin (ThermoFisher). Pictures were taken using Radius software (Version 2.1) with a Xarosa digital camera (EMSIS GmbH).

Prph2^WT/WT and Prph2^KI/KI mice at 1 month of age were perfused with a solution containing 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PB. Then, the eyes were enucleated and fixed for 2 h in the same solution. After rinsing in 0.1 M PB, the cornea, iris, lens, and vitreous body were removed, and the remaining posterior part of the eye was cut into four quadrants. They were postfixed in 1% osmium tetroxide (OsO₄) in 0.1 M PB for 1 h and then gradually dehydrated in increasing concentrations of ethanol and acetone. The pieces were embedded overnight in EPON 812, and then blocks of EPON containing the pieces were made and polymerized at 60 °C overnight. Ultrathin sections were obtained from blocks and contrasted using lead citrate and uranyl acetate. Images were acquired with an FEI Tecnai Spirit BioTwin transmission electron microscope (Thermo Fisher Scientific) using Radius software v2.1 with an Xarosa digital camera (EMSIS GmbH, Münster, Germany).