Tecnai g2 spirit 120 kv transmission electron microscope

KP-EV were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer. 5 μl were adsorbed to Formvar carbon-coated copper grids and contrasted for whole mount negative staining. Samples were observed using the FEI Tecnai G2 Spirit 120 kV Transmission Electron Microscope. Images were captured on the Advanced Microscopy Techniques XR80C CCD Camera System with AMT Image Capture Engine V601.

Imaginal discs were dissected in 4% PFA in PBS and transferred onto coverslips coated with poly‐L‐lysine. Discs were further fixed for 2 h in 4% PFA + 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 (PB) at room temperature. Discs were then postfixed in 1% osmium/1.5% potassium ferrocyanide for 1 h, followed by 1% tannic acid in 0.05 M sodium cacodylate pH 7.4 for 45 min. Coverslips were dehydrated stepwise through ethanol, infiltrated with 50:50 propylene oxide: epon followed by one change of pure resin every 24 h for 7 days and then polymerised at 60°C overnight. Ultrathin sections of ~75 nm were collected using a UCT ultramicrotome (Leica Microsystems UK), post‐stained with lead citrate and viewed using a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI Company) with an SC1000 Orius CCD camera (Gatan UK).

Immuno electron microscopy was performed at the Biocenter Oulu Electron Microscopy Core Facility. Fresh skin samples from adult mouse tail were fixed with paraformaldehyde (4% in 0.1 M phosphate buffer with 2.5 % sucrose, pH 7.4). After fixation, the samples were immersed in sucrose (2.3M), frozen in liquid nitrogen, and sectioned with a Leica EM UC7 cryoultramicrotome (Leica Microsystems, Vienna, Austria). For immunolabeling, sections on Butvar-coated nickel grids were first incubated in 0.1% glycine-PBS for 10 min followed by blocking (serum, 1% BSA in PBS for 5 min). Primary antibodies, anti-EMB (G7.43.1) and anti-MCT1 (AB1286-1, Millipore), were incubated for 45 min and corresponding secondaries, anti-Rat and anti-Chicken IgG (Jackson Immunoresearch Laboratories Inc. Baltimore, PA, USA), for 30 min followed by incubation with protein A conjugated 10 nm gold (Cell Microscopy Core, University Medical Center Utrecht, The Netherlands) for 30 min. 1% BSA in PBS was used in washing steps and dilutions of antibodies and gold conjugates. The grids were stained with neutral uranyl acetate (UA) and coated with 2% methyl cellulose with 0.4 % UA. Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, Eindhoven, The Netherlands) was used for imaging and Quemesa CCD camera (Olympus Soft Imaging Solutions GMBH, Münster, Germany) for capturing the images.

293 T cells were seeded in dishes the day before infection. After 24 h, the cells were incubated with VSV-G pseudotyped HIV-1 and polybrene at a final concentration of 8 μg/ml in the presence or absence of SJP-L-5 at 37 °C. The virus supernatant was removed and replaced with fresh medium 4 h post-viral infection, and SJP-L-5 was maintained at all times. At 4 h and 8 h post infection, the cells were harvested for the TEM assay. Cells were fixed for 12 h at 4 °C in 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), washed, fixed again in aqueous 1 % osmium tetroxide, and embedded in EPON. TEM was performed with a Tecnai G2 Spirit-120 kV transmission electron microscope (FEI, Hillsboro, OR, USA), at 120 kV, on ultrathin sections (80-nm thick) stained with uranyl acetate and lead citrate. 20 cells per field were selected randomly in the control and SJP-L-5 groups. Quantification of total HIV-1 was performed on 20 fields in the control and SJP-L-5 groups.

Immunoelectron microscopy (immuno-EM) was performed at the Biocenter Oulu Electron Microscopy Core Facility as described previously.^{37 (link)} In short, fresh human placental samples from SPTBs and STBs were fixed and cut with a Leica EM UC7 cryoultramicrotome (Leica Microsystems, Vienna, Austria). For immunolabeling, sections of Butvar-coated nickel grids were exposed to primary antibody to HSPA5 (3177, 1:100 dilution; Cell Signaling Technology) and bound antibodies were labeled by incubation with protein A–conjugated 10 nm gold (Cell Microscopy Core, University Medical Center Utrecht, The Netherlands). Controls were prepared by replacing the primary antibody with PBS. To reduce background labeling, endogenous immunoglobulins were blocked using Fab fragments (Goat Anti-Human IgG [H + L]; Jackson ImmunoResearch Europe Ltd, United Kingdom). Samples were incubated with Fab fragments for 30 min after primary blocking step before incubation with primary antibody. Thin sections were examined with a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, Eindhoven, The Netherlands), and images were captured by a Quemesa CCD camera (Olympus Soft Imaging Solutions GMBH, Münster, Germany).

Parasite cultures ~92 hr following rapamycin (or mock) treatment were fixed at 37°C in 8% formaldehyde in 0.2 M phosphate buffer pH 7.4 (PB) for 15 min by adding 1 vol of fixative solution to 1 vol of culture. The cells were pelleted, then further incubated in 2.5% glutaraldehyde, 4% formaldehyde in 0.1 M PB at room temperature for a further 30 min. Cells were washed in 0.1 M PB before being embedded in 4% (w/v) low-melting point agarose in distilled water. The agarose-embedded samples were cut into 1 mm³ blocks, post-fixed in 1% (w/v) OsO₄ and 1.5% (w/v) potassium ferrocyanide for 60 min at 4°C then incubated sequentially in 1% (w/v) tannic acid in 0.05 M PB for 45 min and 1% (w/v) sodium sulphate in 0.05 M PB for 5 min. The samples were washed in water and dehydrated through a graded series of ethanol before being embedded in Epon resin (Taab 812). Blocks were trimmed and ultrathin 70 nm sections cut using a diamond knife on a UC6 Ultramicrotome (Leica Microsystems, Wetzlar, Germany), picked up on 150 hexagonal mesh copper grids and post stained with lead citrate before being imaged using a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI Company, Hillsboro, OR) with an Orius camera (Gatan Inc., Pleasanton, CA).

The embryos were fixed with 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min and embedded in low‐melt agarose. Agarose‐embedded embryos were then prepared and imaged at the Biocenter Electron Microscopy Core Facility (Oulu, Finland). The embryos were postfixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA), dehydrated in acetone, and embedded in Epon LX112 (#21210; Ladd Research Industries Inc., Williston, VT). Hydroxypropyl methacrylate (Sigma, St. Louis, MO) was used in the embedding of β‐galactosidase‐stained embryos to stabilize the X‐gal reaction product (Masahira et al., 2005 (link)). Thin and semi‐thin sections were cut throughout the embryo using a Leica Ultracut UCT microtome (Leica, Wetzlar, Germany), and toluidine blue‐stained semi‐thin sections were used to select thin sections. The thin sections were stained with uranyl acetate and lead citrate and examined using a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, Eindhoven, Netherlands). Images were captured using a Quemesa CCD camera (Olympus Soft Imaging Solutions GMBH, Münster, Germany).