E3000 critical point drying apparatus

B16F10 cells were grown on sterile 5 mm cover slips in 24 well plates. After treatments, the cells on the cover slips were washed with PBS and fixed with 2.5% glutaraldehyde solution containing 4.5% glucose buffered with 75 mM cacodylate buffer (pH 7.2) for 3 h at room temperature. After 3 washes with 100 mM cacodylate buffer, secondary fixation was achieved by the addition of 1% osmium tetroxide buffered with 50 mM cacodylate (pH 7.2) for 3 h at room temperature. Subsequently, the cells were washed with distilled water, dehydrated in an ethanol series (25%, 50%, 75%, 90% and 100%) and then a 1∶1 mixture of ethanol/acetone, and subsequently kept in pure acetone. Finally, critical point drying was performed with CO₂ in an E 3000 critical point drying apparatus (Quorum Technologies, Newhaven). The specimens were mounted on adhesive carbon discs and sputter-coated with gold in a SC7620 Sputter Coater (Quorum Technologies, Newhaven), and images were taken with an EVO40 scanning electron microscope (Carl Zeiss GmbH, Oberkochen) at 20.0 kV. Quantitation of the image surfaces was carried out by using ImageJ. Cell borders were drawn and pixel numbers within the borders were obtained and converted to µm² units. For each sample, measurements were made on 30 cells, and t-tests were then performed.

Two consecutive cryosections (10 μm) of each allograft sample were placed on Thermanox coverslips and perfused with heparinized whole blood as described for the immunofluorescent imaging above. The perfused sections were cut out of the plastic coverslips with scissors, fixed and processed for scanning electron microscopy. Following repeated washes with 100 mmol/L Na-cacodylate pH 7.2 buffer, samples were fixed in 1 v/v% glutaraldehyde for 16 h. The fixed samples were dehydrated in a series of ethanol dilutions (20–96 v/v%), 1:1 mixture of 96 v/v% ethanol/acetone and pure acetone followed by critical point drying with CO2 in E3000 Critical Point Drying Apparatus (Quorum Technologies, Newhaven, UK). The specimens were mounted on adhesive carbon discs, sputter coated with gold in SC7620 Sputter Coater (Quorum Technologies, Newhaven, UK) and images were taken with scanning electron microscope EVO40 (Carl Zeiss GmbH, Oberkochen, Germany) at 5000x magnification from the intima, media and adventitia layer of each cross-section.