Dfc500

Soil samples were collected from a pristine forest in Vietnam and from an oak forest in Ukraine. Nematodes were extracted from soil samples using modified Baermann funnel technique (Southey, 1986 ).
They were heat killed, fixed in 4% formaldehyde (for morphological observations) or in a DESS mixture (Yoder et al., 2006 (link)) (for molecular analyses), transferred to anhydrous glycerol (Seinhorst, 1962 (link)), and mounted on glass slides for microscopic observation. Measurements were performed with a Nikon digital camera on a Nikon Eclipse Ni microscope at the Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST), Vietnam. Observations of morphological diagnostic features were performed in a Leica DM5000B light microscope using the Nomarsky differential interference contrast (DIC) technique. Illustrations were drawn and photographs were taken using a Leica DFC 500 camera on Leica DM5000B microscope at the Museum and Institute of Zoology Polish Academy of Sciences (PAS), Warsaw, Poland. Illustrations were edited by using Adobe Photoshop CC 2018. The spicule terminology is expressed according to Peña-Santiago et al. (2014) (link).

Sections were dried and washed in 0.1 M TBS for 5 min. Sections were heated in 10 mM sodium citrate at 75°C for 30 min and allowed to cool to room temperature. Endogenous peroxidase activity was blocked with incubation in 3% hydrogen peroxide in methanol for 30 min with shaking. Sections were rinsed with running water for 10 min, after which they were placed in blocking buffer (0.1 M TBS and 2% FBS) for 5 min before blocking with 10% NGS for 30 min in 0.1 M TBS with 0.2% Triton-X. This was followed by overnight incubation at 4°C with anti-amyloid precursor protein (APP) (Invitrogen, USA; #36-3900; 1:1000). Sections were washed in 0.1 M TBS, placed in blocking buffer and then incubated in biotinylated goat anti rabbit secondary (Jackson Labs, #111-065-003; 1:250). Sections were washed and placed in blocking buffer again prior to incubation in Elite ABC (Vectastain, CA, USA; #PK-6100) for 30 min. Sections were washed and incubated in DAB (Vectastain, CA, USA; #SK-4100) for 5 min. Sections were washed in running water for 5 min and stained with Hematoxylin for 30 s. Sections were washed, dehydrated (alcohol 70, 80, 95% 2×, 100% 2×, xylene 2×), and coverslipped in Permount. Images were taken with a Leica DFC500 (Leica, Germany) and the number of varicosities were counted with ImageJ.

The type fossil specimens studied are housed in the Key Lab of Insect Evolution and Environmental Changes, the College of Life Sciences, Capital Normal University in Beijing, China. The specimens were examined and photographed, either dry or wetted with 95% ethanol, under Leica MZ 16.5 dissecting microscope (Leica, Wetzlar, Germany) with attached digital camera Leica DFC500. The specimens illustrated with the aid of camera lucida attached to the microscope. The figures are drawn by CorelDraw 12.0 and Adobe Photoshop CS5. Wing venation terminology is basically adapted from Rasnitsyn & Zhang [2 (link)].

At the end of the experimental period, adult of zebrafish have been carefully euthanized by anesthesia with a dose of 0.7 g/l tricaine methane sulfonate (MS-222) buffered. Gills, liver, gut were excised and immediately fixed in 4% formaldehyde (Bio-Optica) in PBS buffered (Phosphate Buffered Saline, Sigma Life Science) at room temperature for 24 h.
Gills were decalcified, prior to processing, with a decalcifier agent (Biodec R, Bio-Optica) for 2 h at room temperature. Histological examinations were performed according to our standard laboratory procedures. All tissues were washed three times with PBS (0.1 M, pH 7.4), gradually dehydrated in ascending alcohols series (35°, 50°, 70°, 95°, absolute ethanol) for 20 min each one and clarified in xylene for 1 h at room temperature, subsequently embedded in paraffin wax (Medite tissue wax 56–58°C). Five μm thick histological sections cut by microtome (Reichert Jung 1150 Autocut) and collected on glass slides (Menzel Gläser, Thermo Scientific). At least 10 slides of each tissue were collected. The sections were stained with Haematoxylin-Eosin (HE) (Bio-Optica) (Brundo et al., 2011 (link)) and observed under optical microscope (Leica DM750) to identify potential morphological alterations. Photographs were produced using an optical microscope (Leica DMLB) equipped with a digital camera (Leica DFC500).

Cells were cultured on glass coverslips for 48 hours, fixed in 4% paraformaldehyde for 30 minutes at room temperature, and then permeabilized with 0.1% Triton X-100 for 20 minutes at room temperature (Sigma, St. Louis, MO). Anti-rabbit Alexa Fluor 488 and antigoat Alexa Fluor 555 purchased from Invitrogen incubated the cells with the reaction dilution of 1:200 for 30 minutes at room temperature, and then 4’, 6-diamidino-2-phenylindole (DAPI) bought from Sigma was added with the reaction dilution of 1:500 for 10 minutes at room temperature. Co-localization of SOX2 and OCT4 was analyzed using a Leica TCS SP5 confocal microscope (Leica TCS SP5, Wetzlar, German). Images were captured with a Leica DFC 500 digital camera and processed with LAS AF software (Leica).