Osmium tetroxide oso4

The preparation of biological specimens for scanning electron microscopy (SEM) observation was carried out at room temperature according to a procedure described previously by Fischer et al. (54 (link)). Treatment and control coupons were fixed with 2.5% glutaraldehyde (catalog number G6257; Sigma-Aldrich) in 0.2 M sodium cacodylate (pH 7.4) (catalog number C0250; Sigma-Aldrich) for 30 min, rinsed with 0.2 M sodium cacodylate, postfixed with 2% osmium tetroxide (OsO₄) (catalog number 75632; Sigma-Aldrich) in 0.2 M sodium cacodylate for 30 min, and rinsed again with 0.2 M sodium cacodylate. Subsequently, the fixed samples were dehydrated with a graded ethanol series (30 to 100% at 10% intervals for 10 min each) and postdried with hexamethyldisilazane (HMDS) (catalog number 440191; Sigma-Aldrich) for 20 min. The dried samples were mounted onto SEM specimen stubs using conductive double-sided carbon tapes (catalog number 77825-12; Electron Microscopy Sciences, Hatfield, PA) and coated with 10 nm of gold (108auto sputter coater; Cressington Scientific Instruments, UK). Electron micrographs were obtained at an acceleration voltage of 2 kV using a Magellan 400 XHR-SEM instrument (FEI Company, Hillsboro, OR) at the Electron Microscopy Facility, University of Massachusetts—Amherst.

The microbial cultures of S. epidermidis and C. parapsilosis after 24-h cultivation under the same conditions as described in previous experiments were prepared according to the standard protocol [37 ,70 (link),71 (link)]. Our samples were fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA), post-fixed in 1% osmium tetroxide (OsO₄; Sigma-Aldrich) and thoroughly but carefully washed by PBS buffer (Sigma-Aldrich). The process of dehydration by ethanol (VWR Chemicals, Leuven, Belgium) series (30%, 50%, 70%, 80%, 90%, 95%, each step 15 min, and three times 100%) prepared the samples for drying. Here two methods of drying are compared. The first was done by hexamethyldisilazane (SPI-Chem, West Chester, PA, USA; HMDS; CAS 999-97-3) which was diluted with acetone (Sigma-Aldrich). Therefore, before applying this treatment it was necessary to replace the ethanol with acetone in four steps with increasing proportion of acetone (ratio ethanol/acetone 2:1; 1:1; 1:2; pure acetone) [37 ,70 (link),71 (link)]. The second methodology for sample drying is the critical point drying (CPD) using CO₂. The samples, prepared by conventional protocols [37 ,70 (link)] were coated by 10 nm of Au before imaging in a VEGA TS 5130MM SEM at the acceleration voltage 10 kV or in a Magellan 400L SEM (Thermo Fisher Scientific, Hillsboro, OR, USA) at 2 kV.

In order to observe the morphology of the grown iPSCs onto the glasses surface, scanning electron microscopy (SEM) was performed. For this aim, the cells-seeded BGs were firstly washed with phosphate-buffered saline (PBS) and then fixed in a series of solutions including 2.5% (v/v) glutaraldehyde (Merck, Germany) in 0.1 M PBS (2 h) and 0.1% (v/v) osmium tetroxide (OsO4) (Sigma-Aldrich, UK) in 0.1 M PBS (30 min). In the next step, the fixed samples were dehydrated using graded acetone series (30, 50, 75, and 100%) and maintained in 100% acetone before freeze-drying (BOC Edwards, Crawley, UK). At the final step, the cell-glass samples were sputter coated with gold and viewed SEM (Tescan, Vega ts5136MM, CZ) at accelerating voltage of 15 keV.

CLEM offers the benefits of combining the wide field images light (or fluorescent) microscopy with the higher resolution images of transmission electron microscopy (TEM) to spot specific cellular structures. In the present study, for the first time, CLEM is used to specifically localize the position of micronuclei stained using DAPI and imaged using confocal microscopy followed by transmission electron microscopy for high resolution imaging (Supplementary Figure S2). Briefly, the embryos were prefixed with 4% PFA and stained with 4 μg/mL DAPI, imaged on LSM 510 Meta Confocal microscope on a gridded coverslip which enables the marking of ROI (region of interest). Post fixation was performed with 1% Osmium tetroxide (OsO4) (Cat No. 75632, Sigma Aldrich, USA) and 1.5% Potassium ferrocyanide (Cat No. P3289, Sigma Aldrich, USA) solution prepared in phosphate buffer (0.1 M, pH 7.4) for 30 min at RT. Serial dehydration (30–100%) was performed with ethanol followed by infiltration and embedding with Epon-Araldite. Transmission electron microscopy was performed with a Tecnai G2 Spirit Bio-TWIN 120 KV Transmission Electron Microscope on 70 nm sections. The marked regions of the confocal images of the DAPI stained embryos were overlaid atop TEM images of the same cells collected from the serial ultrathin section. The overlay is achieved using the Adobe Photoshop version 12.0.

Scanning electron microscopy (SEM) was used to view healthy WB samples, with and without the addition of spike protein. A total of 10 μl WB was placed on a glass coverslip and prepared according to previously published SEM preparation methods [30 (link),31 (link)], starting with washing steps in phosphate-buffered saline (PBS) (pH = 7.4) (Thermo Fisher Scientific, 11594516) for 20 min. Fixation was performed by coating the slides in 4% formaldehyde (FA) for 30 min, followed by washing them in PBS three times. For each wash, the PBS should be left on for 3 min before removing and washing again. Osmium tetroxide (OsO₄) (Sigma–Aldrich, 75632) was added for 15 min and the slides were washed in PBS three-times with 3 min in each once more. The next step was to serially dehydrate the slides with ethanol followed by a drying step using hexamethyldisilazane (HMDS) (Sigma–Aldrich, 379212). Samples were mounted on glass slides and coated with carbon. The slides were viewed on a Zeiss MERLIN FE-SEM with the InLens detector at 1 kV (Carl Zeiss Microscopy, Munich, Germany).