Autosamdri 814 critical point dryer

The palate and base of the oral cavity of P3 male-line female chickens were dissected and fixed in SEM fixative containing 4% PFA, 2.5% glutaraldehyde in 0.1 M PBS over 48 hr at room temperature. The tissue was trimmed and thoroughly rinsed in 0.1 M PBS and processed further into a series of 1% osmium tetroxide (OsO₄), 1% tannic acid and 1% OsO₄ aqueous solutions for 1 hr on ice. The tissues were dehydrated sequentially with ethanol (35%, 50%, 70%, 95% and 100%; three times at each concentration for 2 hr each). Specimens were dried completely using a critical point dryer (Autosamdri-814 Critical Point Dryer, Tousimis Research Corporation, Rockville, MD, USA). The samples were mounted onto SEM stub, sputter coated with gold and photomicrographs were taken using a Zeiss 1450EP scanning electron microscope (Carl Zeiss MicroImaging, Inc., NY, and Oxford Instruments X-Ray Technology, Inc., CA).

The ultrastructure of 3, 6, and 8 mg/mL EndoECM, 8 mg/mL MyoECM, and 8 mg/mL No-DC Endo hydrogels was evaluated using scanning electron microscopy (SEM). Sample processing was performed in the proteomics facility of SCSIE University of Valencia. This proteomics laboratory is a member of Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. Hydrogels were fixed in 2.5% glutaraldehyde in PBS (Sigma Aldrich, grade II, 25%) for 24 h, washed in PBS, and kept in PBS at 4°C. Then, hydrogels were treated with 2% osmium tetroxide for 2 h and dehydrated in a graded series of alcohol (30, 50, 70, 90, 100% ethanol) for 30 min per wash and kept in 100% ethanol overnight at 4°C. Hydrogels were washed 3 additional times in 100% ethanol for 30 min and critical point dried using a Autosamdri^® 814 Critical Point Dryer (Tousimis) with carbon dioxide (CO₂) at high pressure (1200 pound-force per square inch, psi) as the transitional medium and a maximum heating temperature of 40°C. Dried samples were coated with gold-palladium for 2 min using a SC7640 Sputter Coater (Quorum technologies) and imaged with a SEM FEG Hitachi S-4800 (SCSIE University of Valencia, Spain). To analyze fiber diameter, four measurements per three 30.0 k fields per sample were measured using ImageJ software (Schindelin et al., 2012 (link)).