Bal tec cpd 030 critical point dryer

GAS WT and GAS ΔcepA were cultured to mid-log phase (OD₆₀₀ = 0.4) and adjusted with PBS to 2 × 10⁹ CFU/ml, followed by fixation with 2.5% glutaraldehyde for 24 h at 4°C followed by placement on cover glass slide by cytospin at 1000 g for 3 min. Bacterial samples were postfixed with 1% osmium tetroxide followed by dehydration with increasing concentrations of ethanol (70–100%). After critical point drying (Bal Tec CPD 030 Critical point dryer, Leica Biosystems), the samples were coated with platinum using sputter-coating system (Bal Tec SCD 500 Sputter Coater, Leica Biosystems) and then examined with a field emission scanning electron microscopy (Supra 50 VP, Carl Zeiss) using inlens detector.

Systematic sampling strategies were applied for the collection of the research material. Tissue samples for SEM [30 (link)] were taken from apex, body and root parts of the 5 tongues (8 samples from one half of each tongue). Each sample contained lingual papillae to compare with the studied species of Felidae family.
Samples of lingual papillae were placed in 2.0% glutaraldehyde dissolved in 0.1M phosphate buffer at pH 7.4. Further sample analyses were performed based on methodology of Čížek et al. [31 (link)]. SEM samples were dried at the critical point (Bal-tec CPD 030 Critical Point Dryer, Leica Biosystems, Wetzlar, Germany) and then gold-coated (Balzers SCD 040 by current 30 mA for 4 min, Balzers, Lichtenstein). Photographic documentation was obtained using Tescan VEGA TS 5,136 XM (Tescan, s.r.o., Brno-Kohoutovice, Czech Republic) scanning electron microscope in a high vacuum and accelerated voltage 20 kV using an SE detector.
In this publication, all anatomical and histological terms are used based on the terminology of Nomina Anatomica Veterinaria (2017) [32 ] and Nomina Histologica Veterinaria (2017) [33 ].

SEM analysis was conducted on the adaxial side of the third leaf from the apex. Leaves were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (PBS) (pH 7.2) at room temperature. Samples were then dehydrated in acetone series of 50, 70, 90, 96, and 100% (v/v) and dried in a Bal-Tec CPD 030 Critical Point Dryer with liquid CO₂ (Leica Microsystems). The samples were coated with gold in a Polaron SEM coating unit E5100. SEM images were taken with a JEOL 6400 scanning electron microscope at the Microscopy Unit of the Institute of Leiden (The Netherlands).