Samdri 790

The developmental process of inflorescence and floral organogenesis were observed under a stereomicroscope and a scanning electron microscope. The selected plants described above (collected at 5, 10, 15, 18, 21, 24, 27, 30, and 33 DAG) were dissected using a Leica S6D stereomicroscope and fixed in FAA (37% formaldehyde/glacial acetic acid/95% ethanol/dH₂O, 10:5:50:35, v/v) at least over night at room temperature [51 ]. Samples were then dehydrated for 15 min in each of the following concentrations 50%, 70%, and 90% ethanol (1×/ea.) and 100% ethanol (3×) at room temperature [34 (link)]. Critical point drying was obtained (Samdri-790, Tousimis Research Corporation, Rockville, MD, USA) mounted on aluminum stubs and platinum coated. One hundred and five plants were sampled, and their inflorescences observed and imaged using scanning electron microscopy (Hitachi S-3500N, Tokyo, Japan) under high vacuum.
To evaluate the timing of inflorescence development and floral organogenesis, a total of 98 plants were evaluated considering the developmental stage of the oldest inflorescence in the plant. The developmental stage was assigned according to the nine stages proposed herein for inflorescence development in TK.

Coverslips with GBS overnight culture were air-dried, heat fixed, and then subjected to a standard Gram stain protocol. Images were taken using a Zeiss upright microscope with an attached Axiocam Icc3 camera. For electron microscopy, 1mL (10⁷ CFU) of bacterial cells suspended in PBS was fixed in a cocktail of 2% gluteraldehyde and 1% osmium tetroxide in PBS for 10 minutes. The solution was then passed through a 0.4μm polycarbonate filter to collect bacterial cells and rinsed with 4mL of water. In order to dry the samples, the filters were taken through a series of increasing concentrations of ethanol (50, 75, 85, 95, 100%) before being placed in a Tousimis SAMDRI-790 critical point drying machine. The dried filters were mounted onto SEM sample stubs with a piece of double-sided carbon tape before applying a 6nm layer of platinum with a Quorom Q150ts high-resolution coater. Samples were viewed using an FEI FEG450 scanning electron microscope.

To obtain SEM images, cell-seeded pancreatic ECM hydrogels were first fixed in a 2% (v/v) glutaraldehyde solution (Millipore Sigma-Aldrich, Burlington, MA, USA) at day 14 of the culture. Samples were then dehydrated using ethanol, dried using the critical point drying (CPD) method with a Tousimis Samdri 790 machine (Tousimis Research, Rockville, MD, USA) and sputter coated with 6 nm Au-Pd with an EMS 150T ES machine (Quorum Technologies, Sacramento, CA, USA). SEM images were acquired using a Schottky Field Emission Scanning Electron Microscope (Joel JSM 7900F Peabody, MA, USA).

Cryptococcus sp. biofilms were grown on glass coverslips in microtiter plates with RPMI-1621 for 72 h. Coverslips with biofilms were then washed three times with PBS and transferred to another microtiter plate containing 2.5% glutaraldehyde and incubated for 48 h at 4°C. The samples were serially dehydrated in alcohol, fixed in a critical-point drier (Samdri-790; Tousimis, Rockville, MD, USA), coated with gold-palladium (Desk-1; Denton Vacuum, Inc., Cherry Hill, NJ, USA), and viewed with a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope at a voltage of 10.0 kV; the image was captured at 10,000×.

For tissue isolations, anesthetized animals were secured on a Sylgard-coated plate in chilled ASW. Papillae 2–3 mm in length and tentacle tips were excised from the OV and relaxed in MgCl₂ (0.33 M, 10 mM HEPES, pH = 8.0) for three hours prior to fixation. Papillae were fixed in 2.5% glutaraldehyde in 0.1 M PBS at pH 7.6 for 4 hours at room temperature and washed for 2 hours in 2.5% sodium bicarbonate. A secondary fixation was for 3 hours at room temperature in 2% osmium tetroxide in 1.25% sodium bicarbonate. Tissues were next rinsed 3X in distilled water and dehydrated in ethanol. For critical point drying we used the Samdri-790 (Tousimis Research Corporation). After drying, tissues were processed for metal coating on Sputter Coater (SPI Sputter) using the gold/palladium target. SEM analyses and photographs were done on a NeoScope JCM-5000 microscope (JEOL Ltd., Tokyo, Japan). In all, seven groups of oral veil tissues were processed independently through fixation, drying, metal coating and imaging, each containing between 6 and 10 papillae-containing pieces dissected from the oral veils of two healthy adult Pleurobranchaea.