Sample preparation was done as previously described. Briefly, cells were plated on 12mm round coverslips and fixed with 2.5% glutaraldehyde/0.15M sodium phosphate buffer (pH 7.4) at room temperature. Cells were post-fixed with 1% osmium tetroxide with 2% tannic acid in water for 10 minutes and 1% osmium tetroxide in water for 10 minutes. Coverslips were then rehydrated in ethanol and critical point dried using CO2 as transitional solvent (Samdri-795 critical point dryer, Tousimis Research Cop., Rockville, MD). Coverslips were then mounted and coated with 10 nm of gold-palladium alloy (60Au:40Pd, Hummer X Sputter Coater, Anatech USA, Union City, C). A Zeiss Supra 25 FESEM operating at 5kV, with 5mm working distance and 10 µm aperture was used to gather images (Carl Zeiss SMT Inc., Peabody, MA).
Hummer x sputter coater
Manufactured by Anatech
Sourced in United States
The Hummer X Sputter Coater is a laboratory equipment designed for the deposition of thin conductive or dielectric films on various substrates. It utilizes a sputtering process to deposit materials in a controlled environment. The core function of the Hummer X is to provide a reliable and consistent way to coat samples for further analysis or preparation.
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9 protocols using hummer x sputter coater
1
Scanning Electron Microscopy of Cells
2
Preparation of CHO Cells for SEM Imaging
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CHO cells were grown in cell culture flask and on two coverslips for 24h. Cells from flask were detached by trypsin, spun down, resuspended in 2 mL of culture media. 1 mL of resuspended cells was added to one of the coverslips with spread CHO cells (50% confluency) and incubated for 20 min. Another coverslip containing spread CHO cells was treated with trypsin under microscope observation to insure that the cells rounded but did not completely detach. Both coverslips were fixed with a solution of 2.5% glutaraldehyde/HBSS, pH 7.4, for one hour at room temperature. Following three rinses with 0.15M sodium phosphate buffer, pH 7.4 (PB), the cells were post-fixed in 1% osmium tetroxide in PBS for 30 minutes followed by subsequent treatment with 2% tannic acid for 10 minutes and 1% osmium tetroxide in water for 10 minutes. The coverslips were dehydrated with ethanol (30%, 50%, 75%, 100%, 100%), transferred to a Samdri-795 critical point dryer and dried using carbon dioxide as the transitional solvent (Tousimis Research Corporation, Rockville, MD). Coverslips were mounted on aluminum planchets with double-sided carbon adhesive and coated with 10nm of gold-palladium alloy (60Au:40Pd, Hummer X Sputter Coater, Anatech USA, Union City, CA). Images were taken using a Zeiss Supra 25 FESEM operating at 5kV, working distance of 5mm, and 10μm aperture (Carl Zeiss SMT Inc., Peabody, MA).
3
Microstructural Analysis of DTG-Loaded PLGA Implants
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To investigate drug distribution and microstructure of DTG PSIs prepared by phase inversion, PSIs were prepared with varying w/w ratios of PLGA/NMP (1:2, 1:4, 1:6 and 1:8 w/w PLGA/NMP) and varying concentration of DTG (100 and 110 mg/g, 170 mg/g, 200 mg/g and 220 mg/g respectively). To investigate drug distribution, microstructure and physical state of drugs after the micronization step, micronized DTG-PLGA powders were prepared from 1:6 w/w PLGA/(NMP/DMSO, 9:1) (250 mg/g DTG) ISFI solutions using the method described above. The resulting DTG PSIs and micronized DTG-PLGA powders were mounted on an aluminum stub using carbon tape, and sputter coated with 5 nm of gold‑palladium alloy (60:40) (Hummer X Sputter Coater, Anatech USA, Union City, CA). The coated samples were imaged using a scanning electron microscope with an acceleration voltage of 5 kV, 30 μm aperture, and average working distance of 15 mm (Zeiss Supra 25, Carl Zeiss Microscopy, LLC, Thornwood, NY). SEM imaging analysis was also carried out to investigate the effect of compression force on the microstructure of PSIs. PSIs generated with varying compression forces using 1:6 w/w PLGA/(NMP/DMSO, 9:1) formulation with the methods described above were flash-frozen in liquid nitrogen and lyophilized for 24 h prior to SEM imaging analysis.
4
Microstructural Analysis of ISFIs
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The microstructures of ISFIs were analyzed using SEM imaging [16 (link)]. Drug-loaded formulations (30 µL) were individually injected into 200 mL of release medium (0.1 M PBS with 2% solutol, pH 7.4) and incubated for 7 days at 37 °C. ISFIs were subsequently collected, flash-frozen using liquid nitrogen, and lyophilized for 24 h (SP VirTis Advantage XL-70, Warminster, PA, USA). The lyophilized samples were mounted on an aluminum stub using carbon tape, and sputter-coated with 5 nm of gold–palladium alloy (60:40) (Hummer X Sputter Coater, Anatech USA, Union City, CA, USA). The coated samples were then imaged using a Zeiss Supra 25 field emission scanning electron microscope with an acceleration voltage of 5 kV, 30 µm aperture, and an average working distance of 10 mm (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).
5
Ultrastructural Analysis of Ischemic Intestine
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30-minute ischemic neonate and juvenile jejunum were fixed at 0-, 30- and 120-minutes during the experimental recovery period in separate experiments. Mucosa was rinsed briefly with PBS to remove surface debris followed by immersion fixation in 2% paraformaldehyde/2.5% glutaraldehyde/0.15M sodium phosphate buffer, pH 7.4. Specimens were stored in the fixative overnight to several days at 4°C before processing for SEM (Microscopy Services Laboratory, Dept. of Pathology and Laboratory Medicine, UNC, Chapel Hill, NC, USA). After three washes with 0.15M sodium phosphate buffer (PBS), pH 7.4, the samples were post-fixed in 1% osmium tetroxide in PBS for 1-hour and washed in deionized water. The samples were dehydrated in ethanol (30%, 50%, 75%, 100%, 100%), transferred to a Samdri-795 critical point dryer and dried using carbon dioxide as the transitional solvent (Tousimis Research Corporation, Rockville, MD). Tissues were mounted on aluminum planchets using silver paste and coated with 15nm of gold-palladium alloy (60Au:40Pd, Hummer X Sputter Coater, Anatech USA, Union City, CA). Images were taken using a Zeiss Supra 25 FESEM operating at 5kV, using the SE2 detector, 30μm aperture, and a working distance of 10 to 12mm (Carl Zeiss Microscopy, LLC, Peabody, MA).
6
Scanning Electron Microscopy of Wound Biofilms
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A single wound was evaluated per pig on days 2, 4, 7, and 10 postinoculation. The transparent dressings and a 4 mm biopsy punch tissue sample for each animal were fixed in 4% formaldehyde, 1% glutaraldehyde, and 0.1 M PBS. The samples were washed three times using 0.1 M PBS and then postfixed in 1% osmium tetroxide in 0.1 M PBS for 1 h. The samples were dehydrated in a graded series of ethanol solutions and then dried (Critical point dryer, Model 28000; Ladd Research Industries, Burlington, VT). The samples were mounted using a double-sided carbon tape to specimen stubs. They were then ion coated with gold:palladium (30:70) (Hummer X Sputter Coater; Anatech Ltd., Alexandria, VA). The samples were then visualized using an Amray 3600 FE scanning electron microscope (Bedford, MA) operated at a voltage of 3 kV and analyzed by scanning 10 or more 1,000 × magnified fields within the wounded tissue and on the portion of the dressing overlying the wounded area. Photomicrographs representative of the observed bacterial density were taken at 2,500 × magnification, which appears to be a biofilm.
7
Scanning Electron Microscopy of Implant Microstructures
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Implant surface and microstructures were evaluated by scanning electron microscopy. First, implants were prepared by injecting 20 μL of polymer solution into 10 mL of 0.1 M PBS, pH 7.4 at 37 °C. For consistency, these studies were done using the same batch of ISFI formulation (1:2 w/w PLGA/NMP, PLGA MW 27 kDa, 1 mL). At predetermined time points (1, 3, 7, 14, and 30 days post injection), the implants were removed from the bath solution and flash-frozen then fractured over dry ice. Following freeze-fracture, implants were lyophilized for 24 h (SP VirTis Advantage XL-70, Warminster, PA). The lyophilized samples were subsequently mounted on an aluminum stub using carbon tape, and sputter coated with 5 nm of gold-palladium alloy (60:40) (Hummer X Sputter Coater, Anatech USA, Union City, CA). The coated samples were then imaged using a Zeiss Supra 25 field emission scanning electron microscope with an acceleration voltage of 5 kV, 30 µm aperture, and average working distance of 10 mm (Carl Zeiss Microscopy, LLC, Thornwood, NY).
8
SEM Analysis of Micronized Drug Powders
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Scanning Electron Microscopy of Wound Dressings
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Dressings and wound bed tissue were evaluated by scanning electron microscopy (SEM). A representative mouse from each of the three separate experiments in the placebo, 0.1% GaCi BID, and 0.3% GaCi OD treatment groups was sacrificed at 4 h postwounding and on day 7. The transparent dressings and a 4-mm tissue disc for each animal were fixed in 4% formaldehyde, 1% glutaraldehyde, and 0.1 M PBS. The samples were washed three times using 0.1 M PBS and then postfixed in 1% osmium tetroxide in 0.1 M PBS for 1 h. The samples were dehydrated in a graded series of ethanol solutions and then dried (critical point dryer, model 28000; Ladd Research Industries, Burlington, VT). The samples were mounted by double-sided carbon tape to specimen stubs and ion coated with gold/palladium (30:70) (Hummer X sputter coater; Anatech Ltd., Alexandria, VA). The samples were visualized using an Amray 3600 field emission (FE) scanning electron microscope (Bedford, MA) operated at a voltage of 3 kV. Samples were analyzed by scanning 10 or more fields at 1,000× magnification within the wounded tissue and on the portion of the dressing overlying the wounded area. Photomicrographs representative of the observed biofilm density were taken at 2,500× magnification.
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