H 7000 transmission electron microscope

5 × 10⁵ 3T3/OKT3-1, OKT3-2, OKT3-3, OKT3-CD43, OKT3-7, or OKT3-CD44 cells were grown overnight on an Aclar fluoropolymer film (Electron Microscopy Sciences, Hatfield, PA, USA) in a 24-well plate and incubated with 5 × 10⁵ Jurkat T cells for 2 h at 37°C. After washing with PBS, the cells were fixed in a mixture of 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 2 h, and then post-fixed in osmium tetroxide for 1 h. Samples were washed with 0.1 M cacodylate buffer three times (15 min/wash) and then dehydrated in a series of ethanol from 30 to 100%. After dehydration, the samples were placed in acetone and infiltrated with Spurr’s resin. Polymerization of Spurr’s resin was performed by heating to 60°C for 48 h. Ultrathin sections (60–90 nm) were cut on a Reichart-Jung Ultracut E microtome and then mounted on 300 mesh copper grids. The sections were stained in saturated aqueous uranyl acetate and Reynold’s lead citrate, washed with distilled water, and examined on a Hitachi H-7000 transmission electron microscope at 150,000× magnification. Analysis of intercellular distances was performed with Adobe Photoshop CS5 by dividing the conjugation area between two cells into equally spaced sections (11–20) and measuring the distance at each point. Nine to 15 individual cell conjugates were analyzed for each group.

To characterize the ultrastructural morphology of the crypt epithelial spaces of animals exposed to spaceflight conditions, animals were fixed in the LMA at a final concentration of 2.5% glutaraldehyde/2.5% paraformaldehyde solution containing 0.1 M sodium cacodylate with 0.45 M NaCl, pH 7.4 at room temperature. After landing, animals were processed as previously described37 (link). Briefly, animals were rinsed in cacodylate/NaCl buffer, post-fixed for 45 min with a 1% osmium tetraoxide solution, and dehydrated with a standard ethanol gradient. Animals were then infiltrated with propylene oxide and accelerated Spurr resin and finally embedded in Spurr at 60 °C for 24 h. Thin sections were stained with uranyl acetate and lead citrate and visualized with a Hitachi H-7000 transmission electron microscope at the UF Interdisciplinary Center for Biotechnical Research.

For immunogold labeling analysis, developing roots and apical buds were preserved by high-pressure freezing/freeze-substitution techniques as described in [6 (link)]. For immunolabeling, 80 nm thick sections from Lowicryl HM20 resin embedded specimens were placed on formvar-coated gold or nickel slot grids and blocked for 30 min with 2 % (w/v) non-fat dried milk solution in 0.01 M phosphate-buffered saline pH 7.2 containing 0.1 % Tween-20 (PBST). The sections were washed and then incubated with a 10-fold dilution of the primary antibody, anti-PD8, for 2 h at RT. Sections were washed and transferred to a 25-fold dilution of secondary antibody goat anti-rabbit IgG-conjugated to 10 or 15 nm gold particles (Ted Pella, Inc) for 2 h at RT. Sections were washed and then stained with uranyl acetate solution for 2 min and lead citrate for 4 min. All observations were performed using a Hitachi H-7000 transmission electron microscope operated at 80 KV (Hitachi USA, OH).

Cultured cells suspended in fresh medium at 10-fold the density of the culture were placed on a carbon-coated grid and left for 10 min at room temperature. The medium was removed, and the cells on the grid were washed twice with phosphate-buffered saline (PBS; 75-mM sodium phosphate [pH 7.3], 68 mM NaCl) for 1 min each time. The cells were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in PBS for 10 min and washed twice with PBS. The cells were then treated with a primary antibody against P1 adhesin (14 (link)) at a concentration of 3 mg/ml in PBS containing 2% bovine serum albumin (BSA) (PBS-B) and then washed five times with PBS. Subsequently, the cells were treated with 10-fold-diluted gold-labeled secondary antibody (goat antibody labeled with 5-nm colloidal gold; Sigma, St. Louis, MO) in PBS-B for 30 min at room temperature, washed five times with PBS, and stained negatively with 2% (vol/vol) ammonium molybdate. The cells were then observed under an H-7000 transmission electron microscope (Hitachi, Tokyo, Japan) operating at 75 kV, and images captured by a Fast Scan-F214 CCD camera (2 k by 2 k with 14-micron pixel size; TVIPS, Gauting, Germany).

The PTX-mPEG-PLGA NCs were prepared using the double-emulsion method (11 (link)). PTX solution (1.25 mg in 1.25 ml methanol solution; Shanghai Baoman Biotechnology Co., Ltd., Shanghai, China) was emulsified in the PLGA-mPEG solution (25 mg in 1 ml dichloromethane solution) by sonication (200 W, 5 sec-2 sec-15) using an ultrasonic cell disrupter (JY92-ZD, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China). Subsequently, 10 ml F68 aqueous solution (1 mg/ml) was rapidly added to the first emulsion and sonicated (200 W, 5 sec-2 sec-15). The resultant emulsions were stirred to evaporate the dichloromethane and were then lyophilized (EPSILON 2–6D; Martin Christ, Osterode am Harz, Germany). The NC sizes were measured using an H-7000 Transmission Electron Microscope (Hitachi Ltd., Tokyo, Japan). The size distribution and ζ potential of the NPs in aqueous solution was determined using a Nicomp-380ZLS ζ potential analyzer, from Particle Sizing Systems, Inc. (Port Richey, FL, USA). The drug-loading rate was calculated as the ratio of the amount of PTX encapsulated in NCs to the total amount of NCs (3 mg) initially used. The detection of drug-loading rate was completed by the Shanghai Cancer Institute (Shanghai, China).

Frontal cortex, cerebellum, liver, lung, heart and kidney were processed for transmission electron microscopy as previously described (Ruzo et al., 2012a (link)) and visualized with a H-7000 transmission electron microscope (Hitachi).