Scaffolds previously used on the cytocompatibility studies were further removed from the plates and fixated for SEM analysis, based on the Utah State University Biological Sample Fixation protocol. In brief, scaffolds were washed with 0.1 M HEPES (Merck®, PHG0001) 3 times and further fixated in a 2% glutaraldehyde (Merck®, G5882) buffered solution overnight. The fixative was then removed, and scaffolds were washed with HEPES 3 times, 5 min each, under gentle agitation. From this point, samples were subjected to a crescent series of ethanol (50%, 70%, 95% and 99%) for dehydration, 2–3 times for 10 to 15 min each. Following this, scaffolds were soaked in a crescent series of hexamethyldisilazane (HMDS—Alfa Aesar, A15139)-alcohol solution (1:2; 1:1; 2:1) until complete impregnation in a 98% HMDS solution, 3 times for 15 min. Finally, HMDS was removed from the wells and left to evaporate on an air flow chamber overnight. Samples were coated with Au/Pd by sputtering (SPI Module Sputter Coater) and the SEM/EDX exam was performed using a high resolution (Schottky) Environmental Scanning Electron Microscope with X-ray microanalysis and the Electron Backscattered Diffraction analysis was performed in a Quanta 400 FEG ESEM/EDAX Genesis X4M in high vacuum mode.
A15139
Manufactured by Thermo Fisher Scientific
The A15139 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform specific functions in a laboratory setting. The detailed description and intended use of this product are not available.
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3 protocols using a15139
1
Scaffold Fixation and SEM Analysis
2
SEM Analysis of Bioceramic Scaffolds
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Further, seeded scaffolds were fixated for SEM analysis, as described in previous works [31 (link),33 (link)]. Scaffolds were rinsed three times with a 0.1M HEPES (Merck®, PHG0001) buffer solution and left overnight in a fixative solution containing 2% glutaraldehyde (Merck®, G5882). A dehydration crescent alcohol series (50%, 70%, 95% and 99%) was conducted previously to the incorporation of hexamethyldisilazane (HMDS, Alfa Aesar, A15139). Samples were left overnight to evaporate remaining residues of the reagents.
Following, samples were coated with Au/Pd using sputtering (SPI Module Sputter Coater) for SEM analysis with a high resolution (Schottky) environmental scanning electron microscope with x-ray microanalysis and electron backscattered diffraction analysis, Quanta 400 FEG ESEM/EDAX Genesis X4M, in high vacuum mode.
Following, samples were coated with Au/Pd using sputtering (SPI Module Sputter Coater) for SEM analysis with a high resolution (Schottky) environmental scanning electron microscope with x-ray microanalysis and electron backscattered diffraction analysis, Quanta 400 FEG ESEM/EDAX Genesis X4M, in high vacuum mode.
3
Scanning Electron Microscopy of Ciliated Cells
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After the cells were differentiated, they were rinsed with DPBS. Next, 2.5% glutaraldehyde (G6257, Sigma) was added to PBS to fix the cells at room temperature for 1 h. After removing the fixative, the samples were rinsed three times with DPBS. To dehydrate the cell samples, we used ethanol (32221, Sigma) with a concentration gradient from low to high (35%, 70%, 85%, 95%) at each concentration for 10 min. After reacting the samples with 100% ethanol for 20 min, 100% hexamethyldisilazane (A15139, Alfa Aesar) was applied as a desiccant for 5 min. After the reaction, the hexamethyldisilazane was removed, and the samples were evaporated and dried for 24 h. The samples were observed with a high-resolution thermal field emission scanning electron microscope (JSM-7610F, JEOL, Tokyo, Japan) to detect the growth and distribution of ciliated cells after differentiation.
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