For the SEM and EDS analysis the cyanobacterium Limnothrix sp. 37-2-1 was used. Samples were prepared by harvesting cells from the two-week-old culture (control) and from the AgNO3 solution in which the cells were incubated for 48 h. After incubation with the AgNO3, the cells were washed with sterile double-distilled water, centrifuged, and the pellet was fixed in 2% glutaraldehyde in BG11 medium. Fixation was taking place at 4 °C overnight. Cells were washed in distilled water and 50 μl of the cell suspension was placed on 12 mm diameter round glass cover slips (Electron Microscopy Sciences, Washington, PA) previously coated with poly-l -lysine (CAS#25988-63-0, Electron Microscopy Sciences, Hatfield, PA) and kept for 15–20 s at room temperature for cells to adhere. Following adhesion, dehydration was done sequentially with 40, 60, 80 and 100% ethanol. The samples were critical point dried and SEM and EDAX analysis was carried out using scanning electron microscope (JEOL Ltd., Japan).
Round glass coverslip
Manufactured by Electron Microscopy Sciences
Round glass coverslips are flat, circular pieces of glass used as sample holders for microscopy applications. They provide a smooth, transparent surface for mounting and observing specimens under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Scanning electron microscope, by JEOL (1 mentions)
Multi walled carbon nanotubes, by Merck Group (1 mentions)
S 4200 scanning electron microscope, by Hitachi (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Rimadyl, by Zoetis (1 mentions)
Alexa fluor 647 conjugated dextran, by Thermo Fisher Scientific (1 mentions)
6 protocols using round glass coverslip
1
Silver-Induced Cyanobacterial Ultrastructure
2
MWCNT-Benzoyl Peroxide Crosslinked Coatings
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Multi-walled carbon nanotubes were purchased from Sigma Aldrich with the outer wall diameters of 110-170 nm and lengths of 5-9 μm. Nanoparticle suspensions at 1 mg/ml in anhydrous ethyl acetate were dispersed by sonication for 15 minutes. MWCNT to benzoyl peroxide (BP) mass ratios of 1:1, 1:2, and 1:4 were used for initial characterization. All cell studies were performed on the 1:4 ratio samples. Suspensions were sprayed with an airbrush (Iwata HP-CS) onto 12 mm diameter round glass coverslips (Electron Microscopy Sciences). Prior to spraying the coverslips were cleaned with acetone and autoclaved. During the spraying process, the coverslips were heated on a hotplate to ~60 °C to initialize in situ crosslinking and prevent the liquid suspension from accumulating on the surface. Samples were further thermally crosslinked in an oven at 60 °C for 12 hours. Excess BP was removed by heating the coated coverslips at 150 °C for 30 minutes.
3
Platelet Adhesion Assay Protocol
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Platelet adhesion assays similar to those described above were performed with minor changes. Platelets at a concentration of 2×107 platelets/mL were allowed to adhere to substrates (30 µg/mL) bound to round glass coverslips (Electron Microscopy Sciences; 22 mm diameter) for 1 hour at 37°C. Coverslips were washed 3 times with adhesion buffer. Adherent platelets were fixed using 2% glutaraldehyde for 30 minutes at 21°C, washed 3 times with 0.1 M sodium cacodylate buffer and processed (fixed, dried, and sputter coated) in the VUMC Cell Imaging Shared Resource and the EM Core. Imaging was done using a Hitachi S-4200 Scanning Electron Microscope.
4
Fibronectin Coating of Glass Coverslips
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15-mm round glass coverslips (Electron Microscopy Sciences, Cat# 72228-01) were coated in 5 μg/cm2 of fibronectin (Sigma, Cat# F1141) diluted in PBS at room temperature for 5 min. The solution was removed and slides were allowed to dry for at least 45 min before use.
5
Chronic Glass-Covered Cranial Windows for In Vivo Imaging
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Chronic glass-covered cranial windows were implanted as described previously (Mostany and Portera-Cailliau, 2008 (link); Holtmaat et al., 2009 (link)) at least 3 weeks before the beginning of the in vivo imaging of dendritic spines (Figure 1A ). The average age at the time of entering the experiment was 3.5 ± 0.9 months (mean ± SD; range 2.1–4.8 months) and 3.1 ± 0.7 months (range 2.3–4.4 months) for the female and male mice, respectively. Briefly, mice were anesthetized (isoflurane, 5% for induction, 1.5% for maintenance via nose cone) and placed on a stereotaxic frame. Dexamethasone (0.2 mg/kg; MWI/VetOne) and carprofen (Rimadyl® 5 mg/kg; Zoetis, Inc.) were administered subcutaneously to reduce brain edema and local tissue inflammation. A 4 mm-diameter craniotomy was performed with a pneumatic dental drill over the primary somatosensory cortex, 3 mm lateral to the midline and 1.95 mm caudal to Bregma. A round glass coverslip (5 mm #1; Electron Microscopy Sciences) was gently laid over the dura mater, covering the exposed brain and part of the skull and glued to the latter with cyanoacrylate-based glue. A layer of dental acrylic (Lang Dental Mfg. Co., Inc.) was then applied throughout the skull surface and up to the edges of the coverslip. A titanium bar (9.5 × 3.2 × 1.1 mm) was embedded in the dental acrylic to secure the mouse onto the stage of the microscope for imaging.
6
Dextran Uptake in BV2 Microglia
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BV2 microglial cells were plated on 24-well plates with a round glass coverslip (Electron Microscopy Sciences, 72196-12) for 2 days prior to the experiment. The cells were placed on ice for 10 minutes, followed by incubation with Alexa Fluor 647–conjugated dextran (Molecular Probes, Thermo Fisher Scientific, D22914) at 1 mg/mL in DMEM for 20 minutes at 37°C. After washing twice with PBS, cells were fixed with 4% paraformaldehyde (PFA, MilliporeSigma, 158127) for 10 minutes.
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