As an additional method to study cell and tissue integrity, TEM was used to detect morphological changes at the ultrastructural level at 0 h and on duplicate samples (OT-culture IDs 5, 11, and 13) at 24 h and 72 h. Briefly, slices were washed twice with PBS and fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at 4 °C. Slices were post-fixed for 2 h in 2.0% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at 4 °C, subsequently dehydrated in ethanol followed by acetone, and embedded in LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (50–60 nm) from regions containing tumor were cut using a Leica Ultracut UCT/Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT7700 electron microscope (Tokyo, Japan) at 80 kV. Digital images were acquired using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
Ultracut uct em uc 6
Manufactured by Leica
Sourced in Austria
The Ultracut UCT/Leica EM UC 6 is a high-precision ultramicrotome designed for sectioning a wide range of materials, including biological samples, for transmission electron microscopy (TEM) analysis. It features advanced cutting capabilities and a user-friendly interface.
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6 protocols using ultracut uct em uc 6
1
Ultrastructural Analysis of Tumor Tissue
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Ultrastructural Analysis of Au-NP Exposure in THP-1 Cells
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THP-1 cells were exposed for 4 h to freshly dispersed Au-NPs at a final concentration of 50 µg/mL. After exposure, the cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at room temperature for 30 min and further fixed overnight in the refrigerator. Samples were rinsed in 0.1 M phosphate buffer and centrifuged. The pellets were then post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at 4 °C for 2 h, dehydrated in ethanol followed by acetone and embedded in LX-112. Ultrathin sections (approx. 50–60 nm) were cut by using a Leica ultracut UCT/Leica EM UC 6. Sections were contrasted with uranyl acetate followed by lead citrate and examined using a Tecnai 12 Spirit Bio TWIN transmission electron microscope (FEI Company) at 100 kV/Hitachi HT 7700. Digital images were taken using a Veleta camera (Olympus Soft Imaging Solutions).
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Transmission Electron Microscopy of MWCNT-Exposed Cells
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MWCNT-exposed cells were analyzed as described previously (Witasp et al. 2009) (link). Briefly, HMDMs were exposed to MWCNTs for 24 h and PMNs were exposed for 3 h. After exposure, the cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at room temperature for 30 min and further fixed overnight in the refrigerator. Samples were rinsed in 0.1 M phosphate buffer and centrifuged. The pellets were then post-fixed in 2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at 4 C for 2 h, dehydrated in ethanol followed by acetone and embedded in LX-112. Ultrathin sections (50-60 nm) were cut by using a Leica ultracut UCT/Leica EM UC 6. Sections were contrasted with uranyl acetate followed by lead citrate and examined using a Tecnai 12 Spirit Bio TWIN transmission electron microscope (FEI Company) at 100 kV/Hitachi HT 7700. Digital images were taken using a Veleta camera (Olympus Soft Imaging Solutions).
4
Transmission Electron Microscopy of Cells
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Following termination of treatments, cells were washed briefly with PBS and fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at room temperature for 30 min. After fixation, cells were rinsed in 0.1 M phosphate buffer and centrifuged. The pellets were then post-fixed in 2.0% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at 4°C for 2 hour, dehydrated in ethanol followed by acetone and embedded in LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT7700 (Tokyo, Japan) at 80 kV. Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
5
Antimicrobial Effect of PLNC8 αβ on P. gingivalis
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The antimicrobial activity of PLNC8 αβ on P. gingivalis was visualized using the fluorescent dye Sytox® Green, which can only cross damaged membranes and fluoresce upon binding to nucleic acids. P. gingivalis were washed and resuspended in Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, and 10 mM glucose, pH 7.3) and incubated in the presence or absence of PLNC8 αβ in 96-well microtiter plates. Images were captured with Olympus BX41 at 40× magnification.
Transmission electron microscopy (TEM) was used to visualize the damage of P. gingivalis, caused by PLNC8 αβ. Briefly, P. gingivalis ATCC 33277 were pelleted and washed with KRG. The bacteria were then treated with 280 nM of PLNC8 αβ in a molar ratio of 1:2 for 2 min and 10 min, followed by fixation in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Specimens were washed in 0.1 M phosphate buffer, postfixed in 2 % osmium tetroxide in 0.1 M phosphate buffer for 2 h and embedded into LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
Transmission electron microscopy (TEM) was used to visualize the damage of P. gingivalis, caused by PLNC8 αβ. Briefly, P. gingivalis ATCC 33277 were pelleted and washed with KRG. The bacteria were then treated with 280 nM of PLNC8 αβ in a molar ratio of 1:2 for 2 min and 10 min, followed by fixation in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Specimens were washed in 0.1 M phosphate buffer, postfixed in 2 % osmium tetroxide in 0.1 M phosphate buffer for 2 h and embedded into LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
6
Antimicrobial Effects of PLNC8 αβ on P. gingivalis
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The antimicrobial effects of PLNC8 αβ on P. gingivalis were visualised by transmission electron microscopy (TEM). Briefly, viable P. gingivalis ATCC 33277 was centrifuged and the bacterial pellet washed with Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4 and 10 mM glucose, pH 7.3). PLNC8 αβ was added to a final concentration of 2.5 μM (molar ratio of 1:1) for 2 min, followed by fixation in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.3. Samples were washed in 0.1M phosphate buffer and postfixed in 2% osmium tetroxide in 0.1M phosphate buffer for 2 h and embedded into LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (∼50–60 nm) were cut by a Leica ultracut UCT/Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Images were captured using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany). The antimicrobial activity of PLNC8 αβ and scrambled-PLNC8 αβ was determined by using Sytox® Green, which can only penetrate damaged membranes and fluoresce upon binding to nucleic acids.
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