1011 transmission electron microscope

For correlative light and electron microscopy, cells were seeded and imaged on gridded glass-bottom dishes (MatTek). Prior to high resolution time-lapse videomicrosopy, low magnification images of the cells to be examined were captured to obtain their coordinates so that they could be identified during EM processing. Immediately post-acquisition, cells were fixed in 2.5% glutaraldehyde in PBS and processed for flat embedding in resin65 (link). After curing, the resin containing the cells was broken away from the plastic dish. Cells of interest were located by reference to the grid coordinates transferred onto the resin. 60 nm sections were cut parallel to the substratum and imaged after on-grid staining in a Jeol (Tokyo, Japan) 1,011 transmission electron microscope.

Cells were fixed by incubation for 24 h in 4% paraformaldehyde, 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), and then washed in phosphate-buffered saline (PBS) and post-fixed by incubation with 2% osmium tetroxide for 1 h. Samples were then fully dehydrated in a graded series of ethanol solutions followed by a propylene oxide bath. The pre-impregnation step was made by a propylene oxide/Epon resin mixture and finally overnight in pure resin for impregnation of the samples. Cells were then embedded in Epon resin, which was allowed to polymerize for 48 h at 60 °C. Ultra-thin sections (90 nm) of these blocks were obtained with a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Sections were deposited on gold grids and stained with 2% uranyl acetate and 5% lead citrate. Microscopy was performed using a JEOL 1011 transmission electron microscope. Images were analyzed using ImageJ software (NIH). Analyses were performed on 10 cells’ analysis for each section. This was repeated four times.

The hydrodynamic diameter and polydispersity index (PDI) of nanoparticles in aqueous solution (0.5 mg/mL) were determined by dynamic light scattering (DLS) analysis at 25 °C using a NanoBrook Omni Particle Size Analyzer (Brookhaven Instruments Corporation, New York, NY, USA) equipped with a 35-mW red diode laser (nominal wavelength 640 nm). Electrophoretic mobility, i.e., ζ-potential, was measured at 25 °C using the same instrument. The morphology of nanoparticles was analyzed by transmission electron microscopy (TEM): nanoparticles (0.1 mg/mL) were dispensed as a drop on a carbon-coated nickel grid and after 20 min, any excess of the solution was absorbed by filter paper. The nanoformulation was subsequently observed with a Jeol Jem-1011 transmission electron microscope (Jeol Jem, Peabody, MA, USA).

Lungs were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in PBS for 2 hours at RT and overnight at 4°C, postfixed with 1% osmium tetroxide/0,8% potassium ferricyanide for 1 h at 4°C, dehydrated in graded acetone solutions and embedded in Epon-812 (TAAB Laboratories Ltd., Berkshire, UK). Semi-thin sections (1 µm) were obtained with a Leica EM UC6 ultramicrotome and stained with toluidine blue. Images were acquired with a Leica DM2500 microscope (Leica, Wetzlar, Germany). Ultra-thin sections (80 nm) were obtained with a Leica EM UC6 ultramicrotome, counterstained with uranyl acetate and lead citrate and examined with a Jeol 1011 transmission electron microscope (Tokyo, Japan).

For ultrastructural
evaluation, the hydrogels were fixed with 2.5% glutaraldehyde in 0.1
M cacodylate buffer (pH = 7.4) for 1 h at RT and then for 3 h at 4
°C. Afterward, samples were postfixed with 1% osmium tetroxide
in 0.1 M cacodylate buffer for 2 h at 4 °C, dehydrated in an
ethanol series, infiltrated with propylene oxide, and embedded in
Epon resin. Cross sections of each hydrogel were cut to allow for
internal analysis. Ultrathin sections (80 nm thick) were stained with
uranyl acetate and lead citrate (15 min each) and observed with a
Jeol Jem 1011 transmission electron microscope (Jeol Jem, USA), operating
at 100 kV. Images were captured using an Olympus digital camera and
iTEM software. Unstained ultrathin sections were observed with a Zeiss
Libra 120 plus TEM operating at 120 keV and equipped with a Bruker
XFlash 6T-60 SDD detector for energy-dispersive X-ray spectroscopy
(EDX).

A two-electrode setup cell, based on the modified CPE and reference Calomel reference electrode (Hanna-Italy), connected to a Jenway 3310 pH meter of (England), was applied for measurement of different IVR concentrations (10^–8 to 10^–3 M); the cell can be described as follows:
UV-Vis spectroscopic measurements were performed using an OPTIZEN POP-automated UV-Vis spectrophotometer (Korea). MIP incubation with IVR solutions was performed in an Eppendorf Thermomixer comfort (Germany). Shaking was performed by a programmable incubator/mixer (Awareness Technology Inc., United States). Centrifugation was performed using the spectra scientific Merlin-503 centrifuge (England). A JEOL 1011 transmission electron microscope was used for sample imaging (Barhoum and Luisa García-Betancourt, 2018 (link); Barhoum et al., 2018a (link)). A SHIMADZU IR spectrometer was employed for Fourier-transform infrared spectroscopy (FTIR) analysis non-imprinted, MIP, and NIP (Teng et al., 2003 (link); Yan et al., 2013 (link)).