Dfc490 digital camera

For morphological characterization, strains were cultivated on PDA (Difco, USA), Malt Extract Agar (MEA), (Difco, USA) with 10% NaCl (w/v) and Dichloran Glycerol Agar (DG-18), (Sigma-Aldrich, USA) for up to 6 months. Morphological analysis was performed directly on the cultured media plates or using the slide culture technique. Preparations were transferred into slides, observed with a light microscope (Leica DM 4000B (Leica, Germany)), and photographed (Leica DFC 490 digital camera (Leica, Germany)). At least 30 measurements per structure were considered. Representative drawings of microscopic morphological characteristics were obtained with Adobe Illustrator CC (Adobe, USA).

Whole mount in situ hybridization (ISH) was modified after [81 (link)] with the following changes: (1) Template DNA for producing DIG-labelled macIF probe (558 bp) was made using Phusion® polymerase (New England Biolabs) with the primer couple 5′-AAGGAGACTGAGCGAGTGAAGC-3′ and 5′-GGATCCTAATACGACTCACTATAGGCATGACGTCCATCTTGTTGTCG-3′. (2) After hybridization, animals were transferred from the reaction tubes into 24 mesh baskets (53 μm mesh size; INTAVIS Bioanalytical Instruments AG, Germany) which were placed into custom made holes drilled into the lid of a 24-well tissue culture plate. Plates with pre-warmed buffers were prepared and the lid with the baskets and animals was transferred to the successive solution. For colour development animals were moved to a plate without baskets.
For semi-thin sections whole mount in situ hybridizations were slightly overstained and fixed in BOUIN’s fluid for several hours. Specimens were dehydrated in an ethanol series and embedded in PolyBed 812 and polymerized for 48 hours. Specimens were cut serially with 2 μm semi-thin sections using a Reichert Autocut (Reichert, Vienna) and a Diatome Histobutler diamond knife (Diatome, Switzerland). Sections were examined with a Leica DM5000B microscope (Leica, Germany) microscope, a Leica DFC490 digital camera and Leica application suite software.

CCK-8 assay (APExBIO) was used according to the manufacturer’s instructions. The transfected cells were seeded into 96-well plates at 4 × 10³/well. At the indicated time, 10 μl of CCK-8 was added to each well, which contained 90 μl of medium. After incubation for 3 h, the optical densities at 450 nm of each well were measured using a microplate reader (Sunrise). Each sample had four duplicate wells and was independently performed in triplicate.
In EdU incorporation assay, cells were seeded at 5 × 10⁴ cells per well in 24-well plates and cultured for 36 h. Cells were incubated with 10 μM EdU for 4 h before the end of the culture period. Following culture, cells were fixed with 4% PFA, and then detected with Click-iT EdU Imaging Kit (Life Technologies, CA) according to the manufacturer’s procedure (Life Technologies, CA). The plates were visualized under ×20 magnification using Leica DMI 3000B with Leica DFC490 Digital Camera. Quantification of the percentage of EdU-positive cells was performed using the ImageJ program.
For the colony formation assay, 500 cells were plated in a P60 plate and allowed to grow until visible colonies appeared. Colonies were stained with Giemsa and counted.