HSC-3 cells were seeded at 1.5 × 105 cells per well, on fibronectin coated glass coverslips that were placed at the bottom of a 24-well plate. Cells were washed in complete DMEM and stimulated with MPM-1 diluted in complete DMEM to 8.5 μM for 2 or 6 h. One well was left untreated in complete DMEM alone. Processing was performed as described for the transmission electron microscopy samples up until the last step of the dehydration series (100% ethanol). At this point, samples were dehydrated by incubation 3 × 2 min in hexamethyldisilazane (Sigma-Aldrich). The samples were mounted on specimen holders and coated with gold–palladium in a Polaron Sputter Coater (Quorum Technologies, Lewes, UK) before being imaged on a GeminiSEM 360 (Zeiss).
Polaron sputter coater
Manufactured by Quorum Technologies
Sourced in United Kingdom
The Polaron sputter coater is a laboratory equipment used for depositing thin, uniform coatings of conductive materials, such as metals, onto various substrates. It operates by sputtering, a physical vapor deposition process, where atoms or molecules are ejected from a target material and then deposited onto the substrate.
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4 protocols using polaron sputter coater
1
Imaging HSC-3 cells with MPM-1
2
Characterization of Electrospun Nanofiber Scaffolds
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Nanofibers were sputter-coated with a layer of gold approximately 60 nm in thickness using a Polaron sputter coater (SC510; Quorum Technologies, East Grinstead, UK). The samples were examined in an Aquasem (Tescan, Brno, Czech Republic) scanning electron microscope (SEM) in secondary electron mode at 15 kV.
The electrospun scaffolds were characterized in terms of fiber diameter and pore size using mathematical stereological methods, as described in detail in Mickova et al.30 (link) Briefly, the stereological parameters were measured from arbitrarily selected sections of the SEM images, using Ellipse software (version 2001; ViDiTo, Košice, Slovakia). The distribution of the fiber diameters and pore sizes were determined quantitatively from 200 measurements.
The electrospun scaffolds were characterized in terms of fiber diameter and pore size using mathematical stereological methods, as described in detail in Mickova et al.30 (link) Briefly, the stereological parameters were measured from arbitrarily selected sections of the SEM images, using Ellipse software (version 2001; ViDiTo, Košice, Slovakia). The distribution of the fiber diameters and pore sizes were determined quantitatively from 200 measurements.
3
Scanning Electron Microscopy of Inner Ear Hair Cells
4
Microstructural Analysis of FCG Scaffolds
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The microstructure of the FCG scaffolds was examined using scanning electron microscopy (SEM) (Hitachi SU6600 VP-SEM; Hitachi High Technologies America Inc., Clarksburg, USA). To examine the infiltration of fibrin throughout the CG matrix, cross sections at different positions within the FCG scaffolds were formalin-fixed and critical point dried (Quorum E3000 CPD; Quorum Technologies, East Sussex, UK), fixed to an adhesive carbon stub, and then sputter coated with palladium/gold using a Polaron sputter coater (Quorum Technologies). CG matrix and fibrin gels prepared in an identical manner were used as references to compare structural features. SEM imaging was performed at an accelerating voltage of 15kV, utilising the secondary electron detector.
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