Osmium tetroxide

Mitochondrial morphology in myocardial tissues was assessed using transmission electron microscopy (TEM). The tissues were initially fixed in a 3% glutaraldehyde solution and subsequently post-fixed in 1% osmium tetroxide (Sigma-Aldrich). The samples were then dehydrated in an ascending acetone series and embedded in Epon 812 (SPI Supplies, West Chester, PA, USA). Ultrathin sections, with thicknesses ranging from 60–90 nm, were sliced with a diamond knife and stained with uranyl acetate (SPI Supplies) and lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). Images of interfibrillar mitochondria were captured at a magnification of 10,000 × using a JEM-1400-FLASH Transmission Electron Microscope (JOEL, Tokyo, Japan). An investigator, blinded to the experimental conditions, quantified various mitochondrial morphological parameters, such as area, perimeter, and circularity, using the ImageJ software [31 (link)].

BMAT content was analyzed at the distal femur using osmium staining and subsequent µCT analysis (vivaCT40, Scanco Medical, Brüttisellen, Switzerland). Femurs from mice and rats were fixed with 10% PBS-buffered formalin for 24 h and decalcified in Osteosoft (Merck, Darmstadt, Germany). After decalcification, bones were scanned using the vivaCT40 using a standard protocol to ensure complete demineralization. Afterward, bones were incubated with 2% osmium tetroxide (Sigma-Aldrich, Mannheim, Germany) dissolved in 0.1 M sodium cacodylate buffer (Sigma-Aldrich, Mannheim, Germany) for 1 h at room temperature. Specimens were washed and immediately scanned using the µCT with an X-ray energy of 55 keV, 300 ms integration time, and an isotropic voxel size of 10.5 µm. The fat volume (FV) in the rats was calculated from 700 slices using manual contouring and the Scanco bone evaluation software. The threshold was set to 448 mg HA/cm³. In mice, 400 slices were used with a threshold of 180 mg HA/cm³.

Zinc nitrate hexahydrate (98%), dimethylsulfoxide (DMSO, 96%), ascorbic acid (99%), osmium tetroxide (99.8%), glutaraldhyde (50% in water), phosphate-buffered saline (pH 7.4), ethanol (98%), methanol (99.85%), potassium persulfate (98.5%), 2,2-diphenyl-1-picrylhydrazyl (DPPH, 95%) and 2,20 -azino-bis [3-ethyl benzo thiazoline-6-sulphonic acid] (ABST, 98%) were purchased from Sigma-Aldrich (Hamburg, Germany).

Transmission electron microscopy was performed to study the binding affinity of anti‐P. gingivalis to P. gingivalis. P. gingivalis diluted 1:20 in PBS was incubated with anti‐P. gingivalis antibodies diluted 1:100 in PBS. Excess antibodies were washed off with PBS, and gold‐labeled anti‐human IgG was added in the ratio 1:1000 (Nano Gold Probes, NY). The mixture was incubated at 37 °C for 1 h. Excess antibodies were washed with PBS and fixed with 1% osmium tetroxide (Sigma‐Aldrich, Stockholm, Sweden) until mounting on 200 µm mesh formvar‐coated copper grid (Agar Scientific, Stansted, UK). Uranyl acetate (2%, Sigma‐Aldrich, Stockholm, Sweden) was used for staining before examining in JEOL 1230 transmission electron microscope (Department of Pathology, Linkoping University, Sweden).

Cell morphology of the reference strain of C. albicans ATCC 10231 and the FCZ-susceptible isolate of C. auris H0059-13-1421 was evaluated by transmission electron microscopy (TEM) after treatment with peptides PNR20, PNR20-1, and 35409. A suspension of each isolate was prepared in distilled water and adjusted to a concentration of 1–5 × 10⁶ cell/mL. From this inoculum, 500 μL was added to a 1.5 mL Eppendorf tube, mixed with 50 μL of each antimicrobial peptide (100 μM), and incubated at 37 °C for 24 h. Subsequently, cells were harvested and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) and 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed with the same solution and fixed with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, MO, USA), dehydrated by acetone gradient, and resin-embedded. Finally, the cells were stained with uranyl acetate (Thermo Fisher Scientific Inc., Waltham, MA, USA), examined in a transmission electron microscope (JEOL JEM-1400 Plus, Tokyo, TYO, Japan), and photographed with a Gatan camera (Pleasanton, CA, USA) in the pathology laboratory of the Santa Fe Foundation, in Bogotá. Yeasts without treatment with antimicrobial peptides were used as control.

Purified CD3⁻/28-4⁺ IEL-NK cells were fixed using 2.5% Glutaraldehyde (Sigma Aldrich, USA) for 4–6 h. The cells were then submerged in proper animal serum and later sliced into 1 mm³ before another fixation in 2.5% Glutaraldehyde at 4°C for 1–2 h. Next, the cells were washed three times with 0.1 M of sodium Cacodylate Buffer (Sigma Aldrich, USA) for 10 min at 400 × g followed by another fixation with 1% Osmium Tetroxide (Sigma Aldrich, USA) at 4°C for 2 h. The cells were then dehydrated for 10 min each in 35, 50, 75, and 95% acetone percentages and 15 min in 100% acetone. The dehydrated samples were placed in a full resin beam capsule and polymerized in the oven at 60°C for 24–48 h. Later, the samples were cut to 1 μm, stained with Toluidine Blue (Sigma-Aldrich, USA), and then the area of interest was examined using a light microscope. Finally, ultrathin sectioning was performed before the cells were stained with uranyl acetate for 15 min and viewed under the Electron Microscope (TEM) (Hitachi H-7100, Japan).