Lr white resin

Briefly, samples were fixed in 2% PFA and 0.2% GA overnight. After rinsing with phosphate buffer (PB) (0.1 M, pH 7.4), samples were dehydrated through a graded ethanol series (30, 50, 70, 80, 90, and 100%, 10 min each). Samples were infiltrated in a graded mixtures (3:1, 1:1, 1:3) of ethanol and LR White resin (Ted Pella) then changed two to three times pure resin. Finally, samples were embedded in pure resin and polymerized for 48 h at 50°C. The ultrathin sections (70 nm thick) were sectioned with microtome (Leica EM UC6). After rinsing with 0.1 M PB, sections were blocked in 5% goat serum in 1% BSA buffer (with 0.15% Glycine in PB) for 30 min, then incubated with 1:10 diluted Rabbit Anti-mCherry antibody (ab167453, Abcam, Cambridge, United Kingdom) for 2 h. After rinsing with 0.1 M PB six times again, sections were followed by immunogold labeled with 10-nm protein A-gold (1: 50; Cell Microscopy Center, University Medical Center Utrecht, Utrecht, Netherlands) for 1 h. Following rinses with PB, sections were stained with 2% uranyl acetate for 5 min, and imaged by a transmission electron microscope (FEI Tecnai Spirit 120kV).

For histological studies, anthers were isolated from five plants per genotype and treatment on the day before anthesis, fixed in 50 mM Na-cacodylate buffer (pH 7.2) containing 4% (w/v) formaldehyde at room temperature (RT) for 4 h, washed, dehydrated in an ethanol series, and gradually infiltrated with LR white resin (Ted Pella, Redding, CA, United States). The resin was polymerized at 55°C for 48 h. Semi-thin sections (1 μm) were cut using an Ultracut-E microtome (Reichert-Jung, Heidelberg, Germany) and stained with periodic acid-Schiff (PAS) for polysaccharides and with 1% (w/v) Amido Black for proteins. Stained sections were mounted in DPX (Sigma-Aldrich, 100579) and examined under a DMI-6000 microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Amoebic and macrophage culture treated with each NC were centrifuged at 1800 rpm for 5 min and washed 3 times with phosphate buffer solution (PBS, pH 7.4). Cells were then fixed in 4% formaldehyde and 1% glutaraldehyde in PBS by mixing equal volume of fixative and cell suspension. After centrifugation at 1800 rpm for 10 min, the pellet was kept in fresh fixative overnight. Then, the cells were treated 3 times for 15 min with 8% (0.2 M) sucrose in PBS after fixation with 1% OsO4 in PBS for 1 h and rinsed with PBS for 30 min. For dehydration process, ethanol solutions (50, 70, and 95%) were added to the pellets for 15 min each one, absolute ethanol for 15 min twice, and 100% of propylene oxide for 30 min. Infiltration was done with LR white resin (Ted Pella Inc., USA), first adding 1 : 1 LR White: Propylene Oxide for 2 h to the pellets and then stored overnight in 2 : 1 LR White: Propylene Oxide. Samples were embedded in gelatin capsules and baked in 60°C oven for 48 h. Ultrathin sections of 0.5 μm were collected on Formvar/Carbon 200 mesh and Nickel grids and stained with uranyl acetate for 15 min and lead citrate for 3 min.

Sf9-L cells (1.5 × 10⁶) were infected with vAc76:FLAG at an MOI of 10. At the indicated time points p.i., cells were pelleted at 800 × g for 10 min and prepared for IEM with LR White resin (Ted Pella, Inc.) as previously described^{29 (link)}. Immunolabeling was performed with a mouse monoclonal anti-GFP antibody (1:200; Abmart), followed by incubation with goat anti-mouse IgG conjugated to 10-nm gold particles as the secondary antibody (1:50; Sigma). The samples were visualized with a JEOL JEM-1400 TEM at an accelerating voltage of 120 kV.

Human sperm samples were fixed in 2% PFA with 0.2% glutaraldehyde in 0.1M PBS (pH 7.4) at 4 °C overnight. After dehydration in graded ethanol on ice, the samples were penetrated in LR White resin (Ted Pella Inc., USA). Then the samples were polymerized for 5–6 days under ultraviolet light at 4 °C. After that, a glass knife was used to make semi-thin sections (1–2 μm) under a dissection microscope (Motic, China), and the sections were stained with toluidine blue to locate areas of interest. Finally, ultra-thin (70–90 nm) sections, cut with a diamond knife, were collected on 200-mesh nickel grids. To stain the sections, the samples were first blocked in 5% BSA in phosphate buffer solution-tween (PBST) (0.1% Tween 20 in 0.1M PBS) for 1 h, followed by incubation with rabbit anti-human IgG γ chain specific polyclonal antibody (1:100, Dako, Denmark) at 4 °C overnight. After washing, the sections were blocked in PBST for 1 h, and incubated with a secondary antibody for 1 h at room temperature. Then the sections were washed with PBS and distilled water. After the sections were air-dried and stained with 5% uranyl acetate, they were rinsed again in distilled water. Finally, the sections were viewed with a JEOL JEM-1400 transmission electron microscope (TEM) operating at 80 kV.

Parasites were grown on HFF monolayers for 24 h and, subsequently digested with trypsin for collecting samples. Samples were fixed in a 0.1 M sodium cacodylate buffer (pH 7.4) containing 3% (w/v) paraformaldehyde, 0.1% glutaraldehyde (v/v), 4% sucrose (w/v) at 4 °C overnight. The samples were washed four times in a 0.1 M sodium cacodylate buffer containing 4% (w/v) sucrose, before neutralization on ice using a 0.1 M sodium cacodylate buffer containing 4% (w/v) sucrose and 0.1 M glycine, and subsequent dehydration in ethanol. The samples were immersed in LR White resin (Ted Pella, Inc.) at 4 °C overnight, subsequently polymerized at a light intensity of 1.2 × 10⁵ μJ/cm² on ice for 5 h. The LR White imbedded samples were sectioned with a Leica ultramicrotome (EM UC7) at 100 nm and were picked onto 100-mesh formvar-carbon-coated nickel grids. The loaded nickel grids were then blocked and incubated with mouse anti-Ty antibodies (1:5) at RT for 2 h and overnight at 4 °C, followed with washing in PBS. The samples on grids were incubated with Alexa FluorTM 488 goat anti-mouse IgG 10 nm colloidal gold (1:100) for 1 h, followed with washing in PBS and double-distilled water. The samples on grids were stained by 3% phosphotungstic acid, then observed using a JEM1400FLASH transmission electron microscope operation at 120 KV.

To measure the thickness of the mesoglea in healthy and tumorous polyps, individual hydras were relaxed in 2% urethane and fixed with 4% formaldehyde, dehydrated in ethanol and embedded into LR-White resin (Ted Pella, Redding, CA, USA) according to the manufacturer’s instruction. Semi-thin sections (0.5 μm thick) were cut using Ultracut S ultratome, mounted on slides and stained with methylene blue/azur II as described previously [1 (link)]. Average thickness of mesoglea was measured on 10 random locations of each section, and twelve sections for each condition (control and tumor) were evaluated.