Em 912

Transmission electron microscopy (TEM) of samples prepared by chemical fixation was performed with an EM 912 transmission electron microscope (Zeiss) [30 (link)].

Root nodules were pre-fixed in 50 mM PIPES buffer (fixation buffer 1) containing 2.5% glutaraldehyde. The nodules were cut into smaller pieces in this fixation buffer and afterwards transferred to 50 mM cacodylate buffer containing 2 mM MgCl₂ (fixation buffer 2) and 2.5% glutaraldehyde for complete fixation overnight at 4 °C. After washing the samples four times (10, 30, 30, and 50 min) with fixation buffer 2 without glutaraldehyde, post-fixation with 1% osmium tetroxide was carried out for 1.5 h. Afterwards, they were washed again twice with fixation buffer 2 (without glutaraldehyde) and four times with double-distilled water (45, 35, 30, and 30 min). The dehydration of the samples was achieved in a graded acetone series before infiltration and embedding in Spurr’s resin. The thin sections of embedded samples were post-stained with lead citrate for 2 min and investigated on a Zeiss EM 912 transmission electron microscope with an integrated OMEGA filter. The acceleration voltage was set to 80 kV and the images were recorded with a Tröndle 2k×2k slow-scan CCD camera.

HbNPs were characterized by recording their TEM images, UV and visible spectra and FTIR spectra. The size and shape of HbNPs were measured by a high-resolution transmission electron microscope, Zeiss EM 912, at an accelerating voltage of 120 kV. TEM samples of the HbNPs were prepared by placing the product solution on to the carbon-coated copper grids, allowing the solvent to evaporate in air. UV absorbance of HbNPs dispersed in 0.1 M sodium PB (pH:7.0) was recorded in the range from 200 to 600 nm at an interval of 50 nm, using UV spectrophotometer (make: Shimadzu, Japan, model 1700). FTIR spectra of the HbNPs were measured by FTIR spectrometer (Thermo Scientific, U.S.A.) using the standard KBr method over a range of 500–4000 cm⁻¹.

To further confirm the mode of action of celastrol on B. subtilis, transmission electron microscopy (TEM) analysis was performed. Suspensions of B. subtilis in log-phase growth (10⁷ CFU/mL) were treated with celastrol (3 µg/mL) for 1 h at 37 °C and were harvested at 6500× g for 8 min at 4 °C. For comparative purposes, bacterial cells grown under the same conditions were also treated with pristimerin (10 µg/mL). Subsequently, bacteria were washed in fixative buffer, post-fixed in 1% osmium tetroxide in fixative buffer, and washed with distilled water. Sections (1 µm) were cut with a Reichert Ultracut ultramicrotome and stained with toluidine blue; ultra-thin sections were contrasted with uranyl acetate and lead. Preparations were observed under a Zeiss EM 912 transmission electron microscope. Images were captured with a Proscan Slow-scan CCD-Camera for TEM (Proscan, Scheuring, Germany) and Soft Imaging System software (version 5.2, Olympus Soft Imaging Solutions GmbH, Münster, Germany). Control experiments with the same proportion of DMSO were performed in parallel.

ACLAR film slides (Redding, CA, USA) were placed in 24-well plates and 1 × 10⁵ HUVEC were seeded on top in 1 mL media and incubated for 24 h at 37 °C, 5% CO₂, and 95% relative humidity. A volume of 25 µL of a 2 mM PECNP dispersion was added to the media (final dilution 1:40) for 4 h. Incubation was stopped by the addition of a primary fixative (4% formaldehyde,2% glutaraldehyde and 1 mM MgCl₂, in 100 mM sodium phosphate buffer at pH 7.2). Following two post-fixations steps, buffered OsO₄ (1%) and ethanolic uranyl acetate (0.5%), respectively, samples were flat-embedded in epoxy resin (Serva, Heidelberg, Germany) for ultrathin sectioning. Sections were post-stained with uranyl and lead and investigated by TEM (EM912, Carl Zeiss, Oberkochen, Germany).

Synechocystis cells (slr0151^- mutant and wild-type) were harvested in mid-log phase by centrifugation at 5000 g and adjusted to an OD_750nm = 3 in BG-11. Aliquots (2 μl) of the cell suspension were high-pressure frozen at 2100 bar (Leica HPM 100) in HPF gold platelets (Leica Microsystems, Vienna, Austria) and stored in liquid nitrogen (Rachel et al., 2010 (link); Klingl et al., 2011 (link)). The cryofixed cells were then freeze-substituted (Leica EM AFS2) at -90°C with 2% osmium tetroxide and 0.2% uranyl acetate in pure acetone. Freeze substitution was carried out at -90°C for 20 h, -60°C for 8 h, -30°C for 8 h, with a heating time of 1 h between each step, and then held at 0°C for 3 h. Samples were washed three times with pure, ice-cold acetone followed by infiltration with Epon resin (Fluka, Buchs, Switzerland). After polymerization for 72 h at 63°C, ultrathin sections were cut, and post-stained with lead citrate (Reynolds, 1963 (link)). Transmission electron microscopy was carried out at 80 kV either on a Zeiss EM 912 or on a Fei Morgagni 268 electron microscope (FEI). Data analysis was carried out with the Fiji ImageJ software.