Tecnai spirit biotwin

HeLa Kyoto cells expressing AcFL-LAP2ß and H2B-mCherry were transfected with control or PPP1R12A-siRNA and cultured for 56 h. Cells were incubated with 2 μM SiR-actin for 2 h and then fixed in 4% PFA/2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min. After fluorescence imaging, coverslips were processed using the Ellisman NCMIR protocol; post-fixed in 2% osmium tetroxide/1.5% potassium ferrocyanide for 1 h, on ice, then incubated in 1% weight per volume thiocarbohydrazide for 20 min before a second staining with 2% osmium tetroxide, and then incubated overnight in 1% aqueous uranyl acetate at 4 °C. Following this, the cells were stained with Walton’s lead aspartate for 30 min at 60 °C. Cells were dehydrated through an ethanol series on ice, and incubated in a 1:1 propylene oxide/Durcupan resin mixture. Finally, the coverslips were embedded in Durcupan resin according to the manufacturer’s instructions (TAAB Laboratories Equipment) and sectioned (70 nm). Images were acquired using a Tecnai Spirit Biotwin (FEI Company, Eindhoven, Netherlands) transmission electron microscope. Light and electron microscopy overlays were generated using the TurboReg plugin of Fiji software, with an affine transformation. Final adjustment was done by eye in Illustrator CS5.1.

A Hitachi S3400N scanning electron microscope (SEM) equipped with an EDX (Quantex200) was used to study the morphology and chemical composition of the nanopowder samples. Transmission electron microscopy (TEM) images for evaluating particle morphology were taken using an FEI Tecnai Spirit BioTWIN at an operating voltage of 80 kV. To identify functional groups, FTIR spectra were recorded on an FTIR spectrophotometer (Perkin Elmer, Boston, MA, Spectrum 100) in the range of 400 to 4000 cm⁻¹. To perform phase analysis of the nanopowders, X-ray diffraction (XRD) was carried out using an X-ray diffractometer (X’Pert Pro, PANalytical BV, Almelo, The Netherlands) with CuKa radiation source (λ = 1.54056 Å) operated at 40 kV and 30 mA. The average crystallite size was calculated from XRD data using the Debye–Scherrer approximation (Equation (1)).
$D=\frac{\mathrm{K}\mathsf{\lambda }}{\mathsf{\beta }\cos \mathsf{\theta }}$
where β is the full width at half maximum (FWHM) of the peak at the maximum intensity and D is the average crystallite size.
The degree of crystallinity for HAp powder was also calculated according to Equation (2) [30 (link)].
$X_c(\%)=\frac{{\displaystyle \Sigma }{A}_c}{{\displaystyle \Sigma }{A}_c+{\displaystyle \Sigma }{A}_A}\times 100$
where ΣA_C + ΣA_A is the sum of the area under all the HAp and HAp/BG crystalline and amorphous peaks, and ΣA_C is the sum of the areas under the crystalline peaks present in the scan range between 10° and 80°.

MSCs were obtained from compact bones of 1-week-old C57BL/6 male mice [7 (link), 43 (link)]. MSCs from passages 3–5 were used in the experiments. Detailed information was shown in the supplementary materials.
MSCs were cultured in an exosome-free medium (with or without 10 µM GW4869, HY-19363; MedChemExpress, an exosome inhibitor), and exosomes were isolated and purified by ultracentrifugation. Briefly, MSC-conditioned medium was collected after 48 h of culture and centrifuged at 2000×g for 20 min to remove debris and cells. The supernatant was collected and transferred to a new sterile tube and centrifuged at 10,000×g for 15 min. The supernatant was filtered using a 0.22-µm-pore sterile filter, followed by ultracentrifugation at 110,000 × g for 70 min at 4 °C to obtain exosome pellets, which were resuspended in PBS and stored at −80 °C. We used the BCA protein assay kit (UD283191; Thermo Fisher Scientific, Waltham, MA, USA) to determine the protein content of the concentrated exosomes. Exosomes were identified by Western blot (WB) analysis of the marker proteins CD63 and CD81. The morphology of exosomes was observed using a 120 kV refrigerated transmission electron microscope (Tecnai Spirit Bio-TWIN, FEI Company, Hillsboro, OR, USA). The MSC-Exo concentration used in vivo was 8 mg/kg body weight.

The gill fragments were placed into phosphate buffer solution (PBS), that was then replaced by an appropriate volume of 2% OsO₄ in PBS (pH 7.3) and incubated in darkness for 2 h at room temperature. After washing samples with distilled water, they were placed in 2% uranyl acetate overnight and kept in darkness. Next, they were washed again and then dehydrated in a series of ethanol bath solutions, followed by an acetone bath series before resin embedding.
The embedding was performed with resin and, after sectioning into 60 nm slices, the tissues were contrasted with uranyl acetate and lead citrate. The preparation of samples and their analysis were performed at the Centro de Microscopia at the Universidade Federal de Minas Gerais (UFMG), using a Tecnai Spirit BioTwin (FEI Company).

Prophage induction was accomplished by adding 5 μg ml^-1 mitomycin C to exponentially growing cultures (absorbance 0.1) and incubating for four hours. The cultures were centrifuged at 24,000 g for 1 hr at 4°C and the supernatant filtered through 0.45 μm pore diameter Millipore filters. Polyethylene glycol 6000 was added to 10% (w/v) and dissolved. The preparation was incubated at 4°C for 60 min and centrifuged at 8000 g for 10 min at 4°C. The precipitated particles were resuspended in 0.01 times the original culture volume of deionized water, in place of the SM buffer used by Oakey and Owens [119 (link)]. The phages were observed by transmission electron microscopy. Ten microliters of 2% uranyl acetate were deposited on 200 mesh Formvar/Carbon coated copper grids. After 30 sec, 10 μl of phage preparation were mixed with the stain and, after 30 sec, the grids were gently blotted with Whatman paper and allowed to dry for 2–3 min. The grids were observed with an FEI Tecnai Spirit BioTwin transmission electron microscope operated at 80kV transmission electron microscope.