Axio imager 2 light microscope

The morphology of the isolates was assessed according to Landolt 1986 [1 ] as well as Les et al. [11 (link)]. An M205 FCA fluorescence stereo microscope (Leica, Wetzlar, Germany) and Axio Imager 2 light microscope (Zeiss, Jena, Germany) was used.

Adrenal glands were cleaned from surrounding fat and fixed in a 4% paraformaldehyde solution for 1 h. After washing, adrenals were incubated in increasing concentrations (5–30%) of sucrose solution, embedded in Tissue-Tek® O.C.T.™ Compound (Sakura, Japan) and kept at −80°C. For immunofluorescent staining, 6 μm-thick tissue slices were incubated for 1 h in a blocking solution (composed of 0.3% Triton 100 and 5% normal goat serum in PBS) and then incubated overnight at +8°C in a buffer containing antigen-specific primary antibodies (composed of 0.3% Triton 100 and 5% BSA in PBS). The following antibodies were used: polyclonal rabbit antibody anti-mouse thrombomodulin (Thbd, 1:800, ab130152, Abcam), monoclonal rat anti-mouse CD31 antibody (1:100, ab7388, Abcam), monoclonal rat anti-mouse CD34 antibody conjugated with 488 dye (1:50, 11-0341-82, eBioscience) and monoclonal rabbit anti-mouse VCAM-1 antibody (1:200, clone EPR5047, ab134047, Abcam, USA). Primary antibodies were then detected by secondary antibodies conjugated to either Cy3 or 488 fluorescent dyes (VCAM-1, CD31, Thmd1) and counterstained with a nuclear dye (DAPI, 1:10000). Isotype controls were used as controls for CD146 and CD34 antibodies. Pictures were acquired with a × 10 objective using the Axio Imager 2 light microscope (Carl Zeiss, Jena, Germany).

Photographs under incident light were mainly taken with a Zeiss Discovery V20 stereo microscope. Widefield fluorescence images were captured with a Zeiss Axio Imager 2 light microscope combined with a fluorescence imaging system. Confocal images were obtained with a Zeiss LSM710 confocal laser scanning microscope, using the 488 nm Argon laser excitation line. Images under incident light and widefield fluorescence were stacked in Helicon Focus 7.0.2 or Zerene Stacker 1.04. Confocal images were stacked in Helicon Focus 7.0.2. Microtomographic data were obtained with a Zeiss Xradia 520 Versa 3D X-ray microscope at the NIGP micro-CT laboratory and analysed in VGStudio MAX 3.0. Scanning parameters were as follows: isotropic voxel size, 16.916 μm; power, 4 W; acceleration voltage, 50 kV; exposure time, 2 s; projections, 2001. Uncoated specimens of selected species of extant Scirtidae were studied with a S-3400 N Hitachi scanning electron microscope. Images were further processed in Adobe Photoshop CC to adjust brightness and contrast.

For morphology analyses, the ovary and the testis were dissected at 90 and 360 dph. For the early stages (before 90 dph), the abdominal cavity was dripped with the Bouin’s fixation until the gonads were completely covered and turned yellow, and excessive fluid was removed. For histology analyses, the ovary and testis were dissected at 7, 20, 30, 40, 43, 46, 49, 50, 60, 70, 80, 90, 100, 120, 180, 240 and 270 dph. For post-larvae and small juveniles (7–40 dph) in which gonads were too small to be separated, all tissues from the body cavity were included for analysis. For large juveniles and post juveniles, both gonad lobes (40–240 dph) or the large lobes (270 dph) were collected and cut into small pieces (10 mm) for better fixation in Bouin’s solution on a shaker for 24 h at room temperature. After fixation, the tissues were dehydrated through a series of ethanol concentration gradients, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. An Axio Imager 2 light microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) was used to image the stained sections. The sex ratio statistics from 43 to 270 dph were based on histological observation.

The total sperm count and percentage of sperm motility were determined as described previously [20 (link)]. In brief, the distal cauda epididymis was excised and transferred to a 1.5 mL microcentrifuge tube containing normal saline, nicked at two sites with scissors. Spermatozoa were allowed to suspend in pre-warmed normal saline for 5 min at 37 °C. The total sperm count was calculated by counting the number of sperm in 10 squares of the grid after adding two drops of each specimen onto a counting chamber (SEFI-Medical Instruments Ltd., Hicksville, NY, USA) under an Axio Imager 2 light microscope (Carl Zeiss MicroImaging LLC, Goettingen, Germany) at 20× magnification. Sperm count is expressed as 10⁶ sperms/mL. Sperm motility was evaluated under a light microscope at 20× magnification within 10 squares of the grid on a pre-warmed counting chamber. The percentage of motile spermatozoa was analyzed by the formula ((mean number of motile spermatozoa/total number of spermatozoa) × 100) and the results are given as a percentage.