Lr white resin

Cells were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and then post-fixed with 1% OsO₄ for 2 h. Cells were dehydrated using a gradient series of ethanol (30, 50, 70, 90, and 100%). Cell were then incubated with LR White resin (Sigma, 62661) twice for 1 h, and subsequently embedded in LR White resin^{45 (link)}. The solidified blocks were cut into 60-nm sections and stained with uranyl acetate and lead citrate. Samples were observed and imaged under a transmission electron microscope (TEM, Hitachi H-7600; Hitachi High-Technologies Corporation, Tokyo, Japan). Ten fields were selected by the presence of cytoplasm shrinkage, nuclear membrane shrinkage and/ or nuclear chromatin in the outer nuclear layer gathered towards the center with uneven distribution, and the results were averaged.

Yeast cells were sandwiched with the copper disks and frozen in liquid propane at −175 °C. After freeze substitution with 2% glutaraldehyde, 0.5% tannic acid, and 2% distilled water in acetone at −80 °C, the samples were warmed to 4 °C, rinsed with acetone, and fixed with 2% osmium tetroxide in acetone at 4 °C. After dehydration, the samples were infiltrated with propylene oxide, placed into a 70:30 mixture of propylene oxide and resin (Quetol-651; Nisshin EM Co), and embedded. Ultra-thin sections were stained with 2% uranyl acetate, washed, and further stained with lead stain solution (Sigma-Aldrich). Images of cells were obtained using a transmission electron microscope (JEM-1400Plus; JEOL Ltd.) with a CCD camera (VELETA; Olympus Soft Imaging Solutions GmbH).
For immune electron microscopy, HeLa cells transfected with plasmid carrying LETM1-3HA were sandwiched between molybdenum disks, frozen in liquid propane at −175 °C, and freeze-substituted with 0.025% osmium tetroxide and 3% distilled water. After embedding with LR-White resin (Sigma-Aldrich), ultra-thin sections were immunostained with anti-HA monoclonal antibody (Roche, clone 3F10) and gold-conjugated secondary antibody (British BioCell International), and further stained with lead stain solution. Images were collected using the transmission electron microscope JEM-1400Plus with the CCD camera VELETA.

GUS activity detection was performed by immersing the plantlets in a buffer containing potassium ferro- and ferricyanide (0.5 mM each), 50 mM sodium phosphate, 10 mM EDTA, and 0.1% Triton X-100, 10% methanol, and 1 mg/mL X-GLUC (Duchefa). After overnight staining, Arabidopsis plantlets or tissue samples were fixed and embedded in LR White resin (Sigma) following a protocol from Dr. Nicholas Harris (Dept. of Biological Sciences, U. Durham, ftp://ftp.arabidopsis.org/home/tair/Protocols/compleat_guide/2_fix_and_embed.pdf, accessed on 26 July 2022). Resin pieces were sectioned at 2 µm thickness and counterstained with 2% aqueous fuchsine. Light and fluorescence microphotographs were taken using a B-600TiFL microscope (Optika, Ponteranica (BG), Italy), and whole plantlets were photographed under a Stemi 2000-C stereomicroscope (Zeiss, Oberkochen, Germany).

The untreated leaf discs and leaf discs treated with NaCl (300 and 400 mM) were incubated in the fixation solution (2.5% [v/v] glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) under vacuum conditions for 4 h. The leaf discs were then dehydrated in ethanol and embedded in LR White resin (Sigma-Aldrich). Polymerization and ultrathin section (60–80 nm) were obtained as previously described^{31 (link)}. The sections were used for electron microscopy with H-7500 transmission electron microscope (Hitachi, Tokyo, Japan) which was set at 80 kV.

NIH3T3 cells were seeded overnight in 3.5 cm diameter Petri dishes at 2.5 × 10⁶ cell/mL in culture medium. After 24 h, 100 µL of MWCNTs or GFs at 1 mg/mL were added to each well to a final concentration of 50 µg/mL. The cells were then incubated for 24 h at 37 °C under a 5% CO₂ atmosphere. The medium was then removed, and the wells were washed twice with PBS 1× containing Mg²⁺. Next, cells were trypsinized, harvested, centrifuged, and the supernatant was removed. Cells were fixed with 3% formaldehyde for 30 min at room temperature. Subsequently, cells were treated in osmium tetroxide (Sigma-Aldrich, Cat 75632, CA, USA) and dehydrated in a graded series of ethanol. After dehydration, samples were embedded in LR White resin (Cat. L9774, Sigma-Aldrich, USA). Finally, samples were cut using a microtome (ThermoFisher, MD, USA) and loaded into a JEOL 1400 Plus TEM (Jeol, Tokyo, Japan; located at Fundación Santa Fe, Colombia).

Trophozoites from 24 h culture and enriched cyst-like structures were harvested, washed three times with PBS pH 7.4 and centrifuged at 2,000 g, for 5 min. The pelleted cells were re-suspended overnight in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 at 4°C. Next day cells were washed thoroughly with cacodylate buffer and post-fixed for 30 min in 1% osmium tetroxide in cacodylate buffer. The fixed cells were dehydrated in progressive concentrations of ethanol (20, 30, 50, 70, 90, 100%) for 10 min each and embedded in LR White resin (medium grade 707047 Sigma Aldrich) and polymerized at 64°C for 40 h in gelatin capsules. Ultrathin sections of 70 nm were cut using an ultramicrotome, placed on copper grid of 300 mesh (SPI 2030C), contrasted with uranyl acetate and viewed using a transmission electron microscope at 43,000 X magnification.

Small blocks of tissue (0.5×0.5×1 mm) containing secretory cavities at different developmental stages were fixed in a PBS solution (pH 7.2) containing 4% paraformaldehyde and 0.5% glutaraldehyde, and embedded in LR White resin (Sigma). A Leica EM UC7 microtome was used for cutting the sections to a thickness of 70–80 nm.
A nickel grid containing the sections was washed three times with a droplet of PBS-Tween buffer solution for 5 min, and then blocked with 1% BSA for 20 min. The grid was washed three times with PBST for 5 min each and was then floated on PBST containing the anti-CgCaN-specific polyclonal antibodies (primary antibodies, 1:10 v/v) and left to incubate for 3 h at 37 °C. The grid was again washed three times with PBST for 5 min each, then floated on PBST containing colloidal gold antibodies (secondary antibodies, 1:50, v/v; 10-nm gold particles; Sigma-Aldrich) and incubated for 1 h at 37 °C. It was then washed three times with PBST for 5 min each, and twice with d₂H₂O for 5 min each. The grid was then stained with uranyl acetate and lead citrate. Control samples were prepared in a similar manner. In control A, pre-immunization serum was used to replace the primary antibody, and in control B the primary antibody was replaced with PBS. The sections were examined and photographed using a Philips Fei-Tecnai 12 TEM.