Sc7640 sputter coater

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)).

For each experiment at least 15 gemmae were observed for each line. Plants were imaged using a Leica DFC310 FX camera connected to a Leica M165 FC stereomicroscope. Confocal laser scanning microscopy was carried out with the Zeiss LSM510 Meta microscope using the Zeiss Plan-Neofluar 25x/0.8 water immersion lens with Argon/2 laser excitation at 488 nm in order to observe fluorescence emitted by YFP and chlorophyll at 505–550 nm and 645–710 nm, respectively. Fluorescence images were constructed by making maximum intensity projections from a Z-stack containing the epidermal cell layer of gemmae. For scanning electron microscopy (SEM) gemmae collected from gemma cups were immediately fixed in dry methanol, critical point dried using a Tousimis Autosamdri-815, mounted on aluminium stubs and coated with a gold/palladium mixture using a Quorum technologies SC7640 sputter coater. The samples were then imaged with a JEOL JSM-5510 SEM. All processing of confocal microscopy images was carried out using Fiji (Schindelin et al., 2012 (link)). Other images were adjusted using Adobe Photoshop CS4.