Lead citrate

Induced E. coli cells containing recombinant FilP in pET28a plasmid were harvested and fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment) and 4% paraformaldehyde (Thermo Fisher Scientific), embedded in LR white resin (Sigma-Aldrich), and processed with an ultramicrotome (Leica EM UC7) into 70-nm thin sections. Sections were contrasted using uranyl acetate (Polysciences) and lead citrate (Thermo Fisher Scientific). Images were collected with Talos L120 TEM (FEI) using Ceta CMOS detector (FEI).

To analyze the changes in MNPs@SiO₂(RITC)-treated cells, Karnovsky’s fixative solution (Sigma-Aldrich, USA) was used for fixation of MNPs@SiO₂(RITC)-treated BV2 for 12 h at 4 °C. Cells were sequentially washed with 0.1 M cacodylate buffer, pH 7.4, and post-fixed with 1% (v/v) osmium tetroxide (Polysciences, USA) in a 0.1 M cacodylate buffer for 2 h at room temperature. Samples were dehydrated with graded ethanol solutions (50–100%), infiltrated with propylene oxide, and embedded in Epon Mixture (Polysciences, USA). Samples were incubated at 35 °C for 6 h, at 45 °C for 12 h, and at 60 °C for 24 h. Blocks were sectioned with an ultramicrotome (Reichert-Jung, Bayreuth, Germany). Sections were double-stained with 6% uranyl acetate for 20 min (EMS, USA) and lead citrate for 10 min (Thermo Fisher Scientific, USA) for contrast staining. Images were acquired via a SIGMA500 (Zeiss, Germany) transmission electron microscope at the 3D immune system imaging core facility of Ajou University. Particle number, vesicle size, mitochondrial number and size were analyzed using Zen blue 2.3 image analysis module (Zeiss, Germany).

For transmission electron microscopy, morpholino-treated cells were fixed with 2% glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). They were post-fixed with 1% OsO₄ in 0.1 M phosphate buffer (pH 7.4) for 2 h and dehydrated in an ascending graded series (50–100%) of ethanol and infiltrated with propylene oxide. Specimens were embedded using a Poly/Bed 812 kit (Polysciences Inc., Warrington, PA, USA). After embedding, the specimens were polymerized at 65 °C in an electron microscope oven (TD-700; Dosaka EM, Kyoto, Japan) for 24 h. Next, 70-nm-thin sections were double stained with 6% uranyl acetate and lead citrate (Fisher Scientific, Rockford, IL, USA) for contrast staining. Sections were cut with a Leica EM UC-7 (Leica Microsystems) using a diamond knife and transferred to copper grids. All thin sections were observed with a TEM (JEM-1011; JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.

For TEM study, mice were perfused and fixed for 12 h in 0.1M phosphate buffer (PB) followed by 4% PFA containing 2% glutaraldehyde (MERCK, ZC814139734) at 13 weeks of age. They were postfixed with 1% osmium tetroxide dissolved in 0.1 M PB for 2 h and dehydrated inascending gradual series (50–100%) of ethanol and infiltrated with propylene oxide. Specimens were embedded by Poly/Bed 812 kit (Polysciences). After pure fresh resin embedding and polymerization at 65 °C electron microscope oven (TD-700, DOSAKA, Japan) for 24 h. Sections of about 200~250 nm thick section were initially cut and stained with toluidine blue (sigma, T3260) for light microscope. Ultra-thin slices (70 nm) were double stained with 6% uranyl acetate (EMS, 22,400 for 20 min) and lead citrate (fisher, for 10 min) for contrast staining. There sections were cut by LEICA EM UC-7 (Leica Microsystems, Austria) with a diamond knife (Diatome) and transferred on copper and nickel grids. All of the thin sections were observed by transmission electron microscopy (JEM-1011, JEOL, Tokyo, Japan) at the acceleration voltage of 80 kV. For analysis of TEM images, we used gRatio version 3 program in MATALAB the watershed and connectivity theorems to calculate the average g-ratio and the diameter distribution.

TEM was used to observe internalization of the MBNs-NH₂. After 4 hours of incubation with MBNs-NH₂ in a particle:cell ratio of 50 μg/mL of 10⁵ rDPSCs, the rDPSCs were fixed for 12 hours in 2% glutaraldehyde (Merck, Kenilworth, NJ, USA) and paraformaldehyde (Merck) in PBS (Tech&innovation, Chuncheon, Gangwon, Korea), pH 7.4. They were then post-fixed with 1% osmium tetroxide (Polysciences, Warrington, PA, USA), dissolved in PBS for 2 hours, and dehydrated in a gradually ascending series (from 50 to 100%) of ethanol (Merck) and infiltrated with propylene oxide. Specimens were embedded using the Poly/Bed 812 Embedding Kit (Polysciences) according to the manufacturer’s protocol. Sections 200 to 250 nm thick were initially prepared and stained with toluidine blue (Sigma) for observation under a light microscope. Sections of 70-nm thickness were double-stained with 6% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA) and lead citrate (Fisher, Carlsbad, CA, USA) for 20 minutes and 10 minutes, respectively. Stained sections were cut by Leica EM UC7 (Leica Microsystems, Vienna, Austria) with a diamond knife (Diatome) and transferred onto copper and nickel grids. All the sections were observed on TEM (JEM-1011, JEOL) at the acceleration voltage of 80 kV.

For TEM, morpholino‐treated cells were fixed with 2% glutaraldehyde‐2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). They were post‐fixed with 1% OsO₄ in 0.1 M phosphate buffer (pH 7.4) for 2 hr, dehydrated in an ascending graduated series (50%–100%) of ethanol, and infiltrated with propylene oxide. Specimens were then embedded using a Poly/Bed 812 kit (Polysciences, Warrington, PA). After embedding, the specimens were polymerized at 65°C in the electron microscope oven (TD‐700, DOSAKA EM, Kyoto, Japan) for 24 hr. Thin sections (70 nm thickness) were double‐stained with 6% uranyl acetate and lead citrate (Fisher Scientific, Rockford, IL) for contrast staining. The sections were cut using an EM UC‐7 microtome (Leica Microsystems, Seoul, Korea) with a diamond knife and then transferred onto copper grids. All the thin sections were observed by TEM (JEM‐1011, JEOL, Seoul, Korea) at the acceleration voltage of 80 kV.

RPE flatmounts (with the PVDF membrane) were fixed in 2.5% glutaraldehyde (Energy Beam Sciences, East Granby, CT, USA) in 1× PBS (pH 7.4) for 2 h at room temperature and cut to the proper size. The specimens were then washed 3 times with 1× PBS (15 min each wash) and postfixed in 1% osmium tetroxide (Osmium Tetroxide crystals, Electron Microscopy Sciences, Hatfield, PA, USA) with 1% potassium ferricyanide (Fisher Scientific, Pittsburgh, PA, USA) overnight followed by 3 washes in 1x PBS for 15 min each. Samples were then dehydrated in a graded series of ethanol (30%, 50%, 70% in PBS, 90% in H₂O₂, 100% × 3) for 15 min each (30–95%) in reagent alcohol (Fisher Scientific) and 100% requisitioned in pints and then in two 10-min incubations in propylene oxide (Electron Microscopy Sciences). The tissues were infiltrated with a 1:1 mix of propylene oxide and epon (Energy Beam Sciences) overnight followed by incubation with pure epon overnight at 4° C, and further infiltrated with three 1-h changes of epon. Samples were finally embedded in pure epon at 37 °C for 24 h and cured for 48 h at 60 °C. Ultra-thin sections were cut with a Leica EM UC7 Ultramicrotome (Leica, Wetzlar, Germany), stained with uranyl acetate (Electron Microscopy Sciences) and lead citrate (Fisher Scientific), and visualized with a JEM-1400Flash Electron Microscope (JEOL, Tokyo, Japan).