Cm10 tem

Particles were visualized using transmission electron microscopy (TEM). To this end, 10 µl of sample was diluted in water (1:100 v/v), then 10 μl of the diluted sample was mixed with 10 μl of 1% (w/v) uranyl acetate for 30 s. Then, 10 µl of the sample was placed onto a copper grid covered with a Formvar® film (200 mesh) for 30 s. Excess liquid was removed using a filter paper and the grids were dried in a desiccator for at least 24 h. Imaging was performed using Philips TEM CM10 (Eindhoven, Netherlands) fitted with a bottom mounted camera TVIPS TEMCam F416.

Transmission electron microscopy (TEM) was performed to determine ultrastructural and/or morphological alterations in V. vulnificus induced by aBL exposure. In this study, ATCC 27562 was selected as the representative strain, and two aBL exposures of 108 and 216 J/cm² were studied. An untreated sample was used as a negative control. Following the treatments, samples were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde and stored overnight at 4°C. The samples were then centrifuged and washed twice in 0.1 M sodium cacodylate buffer (pH 7.2), and the pellets were post-fixed in 2% ostium tetroxide buffer for 8 h. Samples were then serially dehydrated in ethanol and embedded in resin (Tousimis, USA). Ultrathin sections (<100 nm) were cut by using diamond blades of an ultramicrotome, collected on uncoated 200-mesh copper grids, and stained with uranyl acetate and lead citrate. The images were captured on a Philips CM-10 TEM (Philips Electronics, USA).

Tibialis anterior muscles were dissected and fixed for one hour in a solution containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, immobilised on a Nunc Sylgard coated Petri dish (ThermoFisher Scientific, Waltham, MA, USA) to prevent muscular contraction as previously described [47 (link)]. The muscles were rinsed in the same buffer and dissected further into small blocks that were subsequently processed for transmission electron microscopy (TEM) as described elsewhere [48 (link)]. Briefly, the samples were postfixed with osmium tetroxide (2% in cacodylate buffer), rinsed, en bloc stained with 1% uranyl acetate in 20% ethanol, dehydrated and embedded in epoxy resin (Epon 812; Electron Microscopy Science, Hatfield, PA, USA) that was baked for 48 hours at 67°C. Thin sections were obtained with a Leica ultramicrotome (Reichert Ultracut E and UC7; Leica Microsystems, Wetzlar, Germany) stained with uranyl acetate and lead citrate, and finally examined with a Philips CM10 TEM (Philips, Eindhoven, The Netherlands). Morphometric analysis of mitochondrial cristae complexity was evaluated with a stereological method. Briefly, a regular grid has been superimposed over 10500X TEM micrographs and the number of intersections between the grid and mitochondrial cristae was recorded. The same grid was used for all the different analysis.

MDA-MB-231 wt and GLS shRNA cells were cultured in 10 mm dishes and treated with metformin 30 μM up to 20 days. In addition, in order to reduce autophagic flux, some samples were treated with NH₄Cl 10 mM for the last 17 h in the presence or absence of metformin. Cells were washed with warm PBS and fixed with 2% glutaraldehyde (G7651; Sigma-Aldrich) in 0.1 M sodium cacodylate buffer pH 7.3 (C0250; Sigma-Aldrich) at 4 °C overnight. The following day, samples were collected, washed three times with cacodylate buffer and fixed for 2 h rt with 2% osmium tetroxide (75632; Sigma-Aldrich) in the same buffer. After three washes in distilled water, cells were stained for 15 min at room temperature with 1% uranyl acetate. Samples were then incubated at 45 °C with 3% agarose. After solidification, agarose blocks were dehydrated with ascending acetone concentration. Blocks were embedded in Spurr medium and polymerized overnight at 65 °C. Samples were finally cut in 80-nm sections by a Reighert-Jung Ultra cut E ultramicrotome (Leica Microsystems, Wetzlar, Germany) and picked up on copper grids. The tiny pieces were post-stained in uranyl acetate and bismuth subnitrate and observed in a Philips CM-10 TEM (Fei Italia, Milan, Italy) and micrographs on Kodak 4489 sheet films (Sigma-Aldrich).