Jem 1011 tem

The micelles were prepared by dialysis method [38 (link)]. First, the DS-cad-DXM and DXM were dissolved into the DMSO and then slowly added into the deionized water with vigorous stirring. Finally, the solution was dialyzed for 12 h to obtain the micelles of DXM@DS-cad-DXM. The DXM@DEX-cad-DXM micelles were prepared following a similar method and were used as the control.
The diameter, polydispersity index, and zeta potential of the DXM@DS-cad-DXM micelles and DXM@DEX-cad-DXM micelles were analyzed by using a Particle Analyzer (Delsa Nano C, Beckman Coulter, CA, USA). The morphology of DXM@DS-cad-DXM micelles and DXM@DEX-cad-DXM micelles were determined by using a JEOL JEM-1011 TEM (JEOL-100CXII, Ltd., Tokyo, Japan). The critical micelle concentration (CMC) of DXM@DS-cad-DXM micelles and DXM@DEX-cad-DXM micelles were measured by fluorescence spectroscopy. Furthermore, the stability of the micelles was also studied.
The DXM content in DXM@DEX-cad-DXM micelles consists of two parts: one part is a chemically linked DXM, and the other part is encapsulated by micelles. A certain number of micelles were dissolved into an HCl solution and shaken for 6 h to produce DXM. Then, the drug loading of the DXM was determined by HPLC (Flexar, Perkinelmer, Shelton, CT, USA).

Nickel grids with epoxy-embedded ultra-thin sections (75 nm) were used for antigen retrieval. The sections were immersed in target retrieval solution (TRS; Dako, Glostrup, Denmark) and incubated for 15 min at 110°C, then incubated in blocking solution for 20 min. The sections were then incubated with rabbit polyclonal anti-LC3B antibody (Sigma-Aldrich, St. Louis, Missouri, USA; 1:100 in 0.5% BSA, 0.5 M NaCl, with PBS) for 2 h at 55°C. After washing (0.5% BSA, 0.5 M NaCl, 0.1% gelatin, 0.05% Tween-20 in PBS), the sections were incubated for 2 h at 50°C with goat anti-rabbit IgG-conjugated 10 nm gold particles (Aurion, Wageningen, NL; diluted 1:30 in 0.5% BSA, 0.5 M NaCl, with PBS). After washing (0.5% BSA, 0.5 M NaCl, 0.1% gelatin, 0.05% Tween-20 in PBS), all of the sections were counterstained with uranyl acetate in the usual manner. The sections were examined with a JEM-1011 TEM (JEOL, Tokyo, Japan).

Immunoelectron microscopy experiment was performed as previously described [32 (link)]. UBR4-V5 stably expressing HEK293 cells were fixed in 2% PFA in PBS for 1 h, followed by PBS washing. The fixed cells were collected by scraping and resuspended in 3% gelatin. Solidified gelatin on ice was fixed again with 2% PFA for 15 min. After sequential cryoprotections with 2.3 M sucrose and PVP solution overnight, samples were frozen in liquid nitrogen and were trimmed into 0.5 mm cubes, which were sectioned by cryo-microtome (Leica EM Crion) in 70 nm sections. The primary antibody against V5 was diluted in 0.1 M PBS, supplemented with 0.5% bovine serum albumin (BSA), 0.15% glycine. Secondary antibody, 12nm gold beads labelled donkey anti-rabbit IgG (Jackson ImmunoResearch), was diluted to 1: 25. Sections were incubated with 2.5% glutaraldehyde for 10 min and with 2% Neutral UA acetate for 7 min, followed by incubation in 4% uranyl acetate and methyl cellulose for contrasting and drying. After drying, samples were recorded using JEOL JEM1011 TEM with high resolution AMT digital camera (Peabody, MA).

The size and shape of all silicon, PDMS, and PVA templates were characterized via Scanning Electron Microscope (Helios Nanolab 650). Ultra-high resolution SEM images were acquired at high vacuum conditions after 10 nm aurum coating using a Q150T ES sputter-coater (Quorum). DPNs still embedded within the PVA templates were observed using an A1 confocal microscope (Nikon) equipped with 63 × oil immersion objective. For EM characterization, a DPNs solution was dried on a carbon-copper grid and coated with 10–20 nm of carbon before Transmission Electron Microscope imaging (JEOL JEM 1011 TEM working at 100 KV). The ζ-potential was calculated using DLS (Malvern, UK).

Cells (5.0 × 10⁴/mL) were seeded in cell culture bottles before being treated on the following day with 10 μg/mL of CpG ODN107 for 12 h, followed by treatment with or without irradiation. After incubation for a further 24 h, cells were collected and fixed in cold 2.5% glutaraldehyde in phosphate buffered saline (PBS). The specimens were post-fixed in 1% osmium tetroxide with 0.1% potassium ferricyanide, dehydrated through a graded series of ethanol (30–90%), and embedded in Epon. Ultrathin sections (65 nm) were stained with 2% uranyl acetate and Reynold’s lead citrate, and imaged using a JEOL JEM-1011 TEM at 80 KV. Images were captured using a side-mount AMT 2k digital camera (Advanced Microscopy Techniques, Danvers, MA, USA).