Samdri 795 critical point dryer

The BV-2 cells were cultured on cell-culture-treated coverslips to ensure adhesion. The cells were electrostimulated (Ramp, 100 μA, 20 Hz, 1 h) and were placed in 1/4 Karnovsky’s fixative (1% paraformaldehyde +1.25% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4) for 2 h at room temperature. After fixation, samples were rinsed with 0.1M sodium cacodylate buffer and post-fixed with 1% osmium tetroxide in 0.1M sodium cacodylate buffer for 1 hour. The samples were rinsed in 0.1M sodium cacodylate buffer and distilled water, then dehydrated with graded ethyl alcohol solutions and dried in a Tousimis samdri-795 critical point dryer (Tousimis Research Corporation, MD, USA.) The dried samples were mounted onto an aluminum stub using adhesive carbon tape and sputter-coated with gold using a JEOL DII-29010SCTR Smartcoater (JEOL Unites States Inc., Peabody, MA, USA.) Samples were SEM imaged using a JEOL JCM-7000 Neoscope scanning electron microscope (JEOL Unites States Inc., Peabody, MA, Unites States) at 15 kV with secondary electron image detection for digital TIFF file image acquisition at various magnifications.

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).

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).

The amputated zebrafish caudal fins (n = 3; each group) were put onto microscope glass slides and were bleached with 5% sodium hypochlorite solution (NaOCl; J.T. Baker) in order to remove external tissue and fatty components.⁽³³⁾ The bleaching solution was washed out with deionized water and the fins were dehydrated in ethanol series (50%, 75%, and 100%) for 15 min sequentially. The exposed bones were then separated into proximal and distal regions by performing a section approximately two segments proximal to the cleft. The proximal and distal parts were rapidly frozen under liquid nitrogen and crushed into small particles with mortar and pestle. The still frozen particles were critical‐point‐dried (CPD) in a Samdri‐795 critical point dryer (Tousimis, Rockville, MD, USA) and evaluated. The morphology of proximal and distal regions of the fins were observed using a Scanning Electron Microscope (SEM) SU8010 (Hitachi, Chiyoda, Japan) equipped with an Energy‐Dispersive X‐ray Spectroscope (EDS). The Calcium (Ca) and Phosphorus (P) atomic percentages, were recorded in five different sites using EDS. The areas of analysis were fixed to 7 × 7 μm² and the equipment voltage was set to 15 kV for both characterization methods.

To visualize the vasculature in native and decellularized flaps, a contrast agent (Visipaque, GE Healthcare, Chicago, IL, USA) mixed with normal saline (1:2) was injected into the arterial pedicle using constant syringe pressure. Image acquisition was performed with a Powermobil C-Arm (Siemens, Munich, Germany). Images were exported in DICOM format and visualized with Osirix software 12.0 (Pixmeo, Bernex, Switzerland). This examination was performed on each flap before and after decellularization.
Scanning electron microscopy was performed for decellularized flaps at the Schepens Eye Institute core facility, supported by the NIH National Eye Institute Core Grant #P30EY003790. Briefly, the samples were dehydrated in graded ethanol solutions and dried at the critical point using a Samdri 795 critical point dryer (Tousimis, Rockville, MD, USA), then mounted on aluminum pedestals and chromed using a Gatan high-resolution ion beam coater (Gatan Inc., Pleasanton, CA, USA). Different surfaces of the samples were imaged using a JEOL JSM-7401F field emission scanning electron microscope (JEOL Inc., Peabody, MA, USA), allowing a qualitative assessment of the scaffold architecture.