Jem 1400

The surface morphology of the PR-NLCs was evaluated by TEM (JEM-1400, JEOL, Tokyo, Japan). The PR-NLCs were diluted with distilled water to observe appropriate images. A drop of sample was placed on a formvar/carbon-coated copper grid (Ted Pella, Redding, CA, USA) and allowed to evaporate under ambient conditions. Then, the samples were negatively stained with 2% (w/w) uranyl acetate and air-dried at room temperature for 2 min. The excessive fluid was removed using filter paper. The samples were observed with a JEM-1400 operating at 120 kV and the data were analyzed with the JEOL Simple Measure program (JEOL, Tokyo, Japan).

The cellular uptake and distribution of GQDs-MSNs nanocomposites in cells was further analyzed by TEM (Jeol JEM-1400) according to Graham and Orenstein (Graham and Orenstein 2007 (link)) procedure with minor modification. Briefly, HeLa cells previously incubated with 100 μg/ml of GQDs or GQDs/MSNs nanocomposites for 4 h were fixed with 2.5% glutaraldehyde solution for 2 h and then postfixed in 1% osmium tetroxide for 1 h at room temperature. Then, samples were dehydrated through a graded series of ethanol concentrations (50, 70, 80, 90, 96, and 100%) and embedded in epoxy resin. Ultrathin sections were prepared using ultramicrotome (RMC PowerTome PT-XL), collected onto TEM grids, stained with 1% uranyl acetate, and imaged under Jeol JEM-1400 TEM.

After gelation in glass containers, hydrogels were lyophilized. The samples were cut into pieces, and the microstructures were observed by SEM (JEOLJEM-1400, Japan) at an acceleration voltage of 20.00 kV. DPSCs were directly seeded and cultured on the surface of CS/β-GP hydrogels. After 24 hours, cell-seeded gels were washed with phosphate buffered saline (PBS) for 3 times and fixed with 2.5% glutaraldehyde at room temperature for 4 hours. Then, the hydrogels were dehydrated in a graded series of ethanol (30%, 50%, 75%, 85%, 95%, and 100%) for 15 minutes in each concentration and air-dried overnight to be analyzed by SEM (JEOLJEM-1400, Japan).

TEM analysis was carried out by resuspending mice PRP to an equal volume of 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4., overnight. Then samples were centrifuged at 1000× g for 10 min, washed, and post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer for 60 min at 4 °C. After that they were dehydrated through a graded ethanol series and embedded in resin. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEM 1400 JEOL, Tokyo, Japan).

To evaluate the textural characteristics of the materials and verify their suitability for this study, they were characterized by nitrogen adsorption-desorption using a Micromeritics 3 Flex (Norcross, GA, USA) that allows the specific surface, S_BET, to be obtained by the Brunauer–Emmett–Teller method (BET) [48 (link)] and pore size distribution using the Barret–Joyner–Halenda method (BJH) [49 (link)]. The structure of the nanoparticles could be analyzed using transmission electron microscope (TEM) micrographs in a JEM-1400 JEOL operating between 40 and 120 kV (Tokyo, Japan). Energy dispersive X-ray fluorescence (EDS) was carried out with a CCD camera (KeenVIEW) operating at 120 kV in combination with TEM to determine the atomic composition of the samples. The intensity of the static electric field of the nanoparticles was determined using a Zetasizer Nano-ZS dynamic light scattering device (DLS) (Malvern Instruments, Malvern, UK).

Using an immunogold immunohistochemistry protocol tissues were prepared for TEM [38 (link),39 (link),40 (link)]. The fixation of the samples was carried out with 4% paraformaldehyde in PBS, after which samples were washed in PBS. Samples were cut with a vibratome (Vibratome Series 1000, Pelco 101, Ted Pella, Inc., Redding, CA, USA) into 20 µm thick sections. After washing in PBS, sections were incubated first in 50% ethanol for permeabilization and then in primary antibody for 48 h at +4 °C: rabbit anti-Cx37/GJA4 (1:100, ab181701, Abcam, Cambridge, UK); rabbit anti-Cx40/GJA5 (1:100, ab213688, Abcam, Cambridge, UK); and goat anti-Cx43/GJA1 (1:300, ab87645, Abcam, Cambridge, UK). Next, sections were rinsed in PBS after which overnight incubation followed with gold-conjugated donkey anti-rabbit or anti-goat secondary antibody (1:1000, 711-205-152 and 705-185-147, both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The size of the gold particles used was 12 nm for anti-rabbit and 4 nm for anti-goat antibodies. On the next day, sections were rinsed in PBS, post-fixed in 1% osmium tetroxide (1 h), and then dehydrated in ethanol and embedded in Durcupan ACM resin (Sigma-Aldrich Inc., St. Louis, Missouri, USA). The sections were observed with a transmission electron microscope (JEM JEOL 1400, Jeol Ltd., Tokyo, Japan).