Leo 912ab tem

Images of organoid cultures were captured using a Keyence BZ-X800 microscope with BZ-X800 Viewer software, v01.02.03.02 or an EVOS FL Auto System (ThermoFisher). Size and morphology of organoids was analyzed using OrganoSeg software^{52 (link)} and phase contrast images. Measurements were performed on digital images by blinded investigators using ImageJ software. For histological analyses, organoids were recovered from the culture plates and treated with Histogel (ThermoFisher) prior to formalin fixation and paraffin embedding, following standard protocols. Slides were stained with hematoxylin/eosin and with Alcian Blue to visualize mucus production. TEM was performed as previously described for human organoids^{87 (link)}. Briefly, organoids were fixed in 3% glutaraldehyde, processed for ultrathin sectioning, and then were imaged on a Zeiss LEO 912AB TEM.

The morphological characteristics of the CNCs were evaluated via transmission electron microscopy (TEM), using a LEO 912AB TEM with an Omega energy filter (Zeiss, Oberkochen, Germany) at an accelerating voltage of 120 kV. For the microscopic observations, drops of dilute aqueous suspension of CNCs (≈4 wt %), previously sonicated for 1 min, were deposited on carbon-coated electron microscope grids, negatively stained with 2% uranyl acetate, and allowed to dry. Representative micrographs were selected for measuring the diameter (D), length (L), and aspect ratio (L/D) of the nanocrystals by digital image analysis (iTEM, software, Olympus Soft Imaging Solutions GmbH, Münster, Germany). In addition, the dimensions of CNCs from all heating programs, in dilute suspensions (at pH 8), were also investigated by dynamic light scattering (DLS) measurements (mod. Litesizer500, Anton Paar, Graz, Austria), performed at 25.0 ± 0.1 °C with a 35 mW laser diode light (λ = 658 nm), and collecting the scattered light at 15° and 90°. By applying correlation analysis and the Stokes–Einstein relation, the equivalent hydrodynamic diameters (D_Hy), the polydispersity index (PDI), and size distributions of the scatters were calculated. Four runs were performed, withdrawing three different aliquots for each set of experimental conditions.

WT and SOCS3-null AS-M.5 cells were seeded at a density of 1 × 10⁶ cells per ml into 6-well plates and onto Thermanox coverslips (13 mm diameter) for culturing to confluency. The cells were then fixed in 1.5% (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C for 1 h. Following three washes in 0.1 M sodium cacodylate buffer in 2% (w/v) sucrose, the cells were incubated with 1% (w/v) osmium tetroxide/0.1 M sodium cacodylate for 1 h, washed three times in distilled water, and incubated in 0.5% (w/v) uranyl acetate in the dark for 1 h. Following two rinses in distilled water, cells were dehydrated in stepwise alcohol increments (30–100% (v/v)) and incubated overnight in a 1:1 mix of propylene oxide/TAAB araldite Epon 812 resin. The propylene oxide was then allowed to evaporate to leave pure resin, which was changed twice before the sample was embedded in fresh resin which was allowed to polymerise at 60 °C for 48 h. Ultrathin sections were cut using a Leica Ultracut UCT and a Diatome diamond knife, contrast stained with aqueous 2% (w/v) methanolic uranyl acetate and Reynolds lead citrate, and viewed using a LEO 912AB TEM (Carl Zeiss) at an accelerating voltage of 120 kV. TIF images were captured using an Olympus Soft Imaging System and image contrast modified using Adobe Photoshop CS.