Veleta tem ccd camera

We conducted preliminary transmission electron microscopy (TEM) observation of Barthelona sp. strain PAP020, focusing on the MROs. Cultivated cells were centrifuged and fixed for 1 h at room temperature with a mixture of 2% (v/v) glutaraldehyde, 0.1 M sucrose and 0.1 M sodium cacodylate buffer (pH 7.2, SCB). Fixed cells were washed with 0.2 M SCB three times. Cells were post-fixed with 1% (v/v) OsO₄ with 0.1 M SCB for 1 h at 4°C, then washed with 0.2 M SCB two times. Dehydration was performed using a graded series of 30–100% ethanol (v/v). After dehydration, cells were placed in a 1 : 1 mixture of 100% ethanol and acetone for 10 min and acetone for 10 min for two cycles. Resin replacement was performed by a 1 : 1 mixture of acetone and Agar Low Viscosity Resin R1078 (Agar Scientific Ltd, Stansted, UK) for 30 min and resin for 2 h. Resin was polymerized by heating at 60°C for 8 h. Ultrathin sections were prepared on a Reichert Ultracut S ultramicrotome (Leica, Wetzlar, Germany), double stained with 2% (w/v) uranyl acetate and lead citrate [38 (link),39 (link)], and observed using a Hitachi H-7650 electron microscope (Hitachi High-Technologies Corp., Tokyo, Japan) equipped with a Veleta TEM CCD camera (Olympus Soft Imaging System, Münster, Germany).

To perform the immunodetection of CD133 in ultrathin sections, the cells grown on coverslips were rinsed with PBS and fixed in 3% paraformaldehyde (Sigma-Aldrich) and 0.1% glutaraldehyde (AppliChem GmbH, Darmstadt, Germany) in PBS at room temperature for 60 min. Following a PBS rinse and dehydration, the cells were embedded in LR White medium (Polysciences Inc., London, UK). The labeling of the ultrathin sections was performed on grids. CD133 was detected using mouse monoclonal anti-CD133 antibody (dilution 1:25; Millipore) and a gold particle-conjugated secondary antibody (anti-mouse IgG 20 nm gold, Cat. no. ab27242, dilution 1:40; Abcam). Ultrathin sections incubated without primary antibody or with the TU-01 primary monoclonal antibody against α-tubulin (dilution 1:200; Exbio) were used as controls. Following immunodetection, the specimens were contrasted with 2.5% uranyl acetate (PLIVA-Lachema, Brno, Czech Republic) for 10 min and with Reynolds solution (Sigma-Aldrich) for 6 min at room temperature. The specimens were then observed under a Morgagni 268(D) transmission electron microscope (FEI Co., Hillsboro, OR, USA). The images were captured using an Olympus Veleta TEM CCD camera and analyzed using iTEM Olympus Soft Imaging Solution (Olympus).

Unmated females at 5 days post-eclosion were dissected in ultrapure water (Milli-Q; Sigma-Aldrich). The dissected guts were fixed in a mixture of 2% paraformaldehyde, 2.5% glutaraldehyde and 0.1 mol l⁻¹ cacodylate (pH 7.4) for 1 h at room temperature. After fixation, the guts were washed in 0.1 mol l⁻¹ cacodylate buffer and post-fixed in 1% osmium tetroxide with 0.1 mol l⁻¹ cacodylate buffer (pH 7.4) for 1 h at room temperature. The guts were washed with distilled water and stained with 0.5% uranyl acetate for 2 h at room temperature. After three washes with distilled water, the guts were dehydrated in a graded series of ethanol solutions (65%, 75%, 85%, 95% and 99.5%) and transferred to 100% ethanol. The guts were then soaked in propylene oxide, transferred to a 1:1 mixture of propylene oxide and Quetol 812 resin (Nisshin-EM, Tokyo, Japan), and embedded in Epon 812 resin. Ultrathin sections of approximately 60 nm thickness were collected on copper grids, stained with 0.5% uranyl acetate and then stained with a lead solution containing 1% lead citrate, 1% lead nitrate and 2% sodium citrate (Sato, 1968 ). The sections were washed with distilled water and dried. The sections were observed using a JEM-1010 electron microscope (JEOL, Tokyo, Japan) equipped with a Veleta TEM CCD camera (Olympus, Tokyo, Japan) at an accelerating voltage of 80 kV.