Eclipse ni u microscope

The in situ hybridization on cryosections of presumptive ovaries were performed as previous described [56 (link)]. Sense and anti-sense digoxigenin-labeled cRNAs of fancl were synthesized and used in this study. The cDNA fragment of 786 nt containing the PHD domain of fancl was used to synthesize probe as previously described [38 (link)]. The in situ hybridization were photographed using a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). Staining intensity in germ cells were quantified by analyzing the gray values using Image J software (National Institutes of Health, Bethesda, MD, USA). There were three independent replicates for the fish of each genotype.

After isolation, midguts were fixed in 4% paraformaldehyde in PBS overnight at 4°C. Specimens were then dehydrated in ascending ethanol series and embedded in paraffin. Sections (7 μm-thick), obtained with microtome Jung Multicut 2045, were deparaffinized, rehydrated, and pre-incubated for 30 min with PBS containing 2% bovine serum albumin (BSA) before incubation with anti-H⁺ V-ATPase antibody (Ab 353-2, polyclonal antibody raised against the highly purified V₁ complex of the H⁺ V-ATPase from Manduca sexta (Huss, 2001 ), a gift by Prof. H. Wieczorek, University of Osnabrück, Germany, dilution 1:10000 in 2% PBS/BSA), for 1 h at room temperature. After washing with PBS, sections were incubated for 1 h at room temperature with an anti-guinea pig Cy2-conjugated secondary antibody (dilution 1:200 in 2% PBS/BSA, Jackson ImmunoResearch, West Grove, PA, United States). After washes with PBS, sections were incubated with DAPI (100 ng/ml in PBS) for nuclear staining and then washed. Slides were mounted in Citifluor (Citifluor Ltd, London, United Kingdom) with coverslips and analyzed with an Eclipse Ni-U microscope (Nikon) equipped with TrueChrome II S digital camera (Tucsen Photonics). The primary antibody was omitted in controls.

Following perfusion and fixation of the heart, tissues to be used for immunostaining underwent brief postfixation (1 h at 4 °C) and dehydration with sucrose, before frozen sectioning at a thickness of 14 μm. After blocking with normal donkey serum (ASL050, Acmec biochemical, Shanghai, China), sections were incubated overnight at 4 °C with primary antibodies. Sections were washed 3 times, incubated with the appropriate fluorescent secondary antibodies (1:500; Abcam (Cambridge, UK) and Thermo Fisher Scientific (Waltham, MA, USA)) for 2 h at 25 °C, washed thrice, stained with 4′, 6-diamidino-2-phenylindole (DAPI, C02-04002, Bioss, Woburn, MA, USA), and mounted with antifading mounting medium (S2100, Solarbio, Beijing, China) and microscope cover glass (10212450C, CITOTEST, Rubano, Italy). Images were taken using a Zeiss LSM980 confocal microscope and a Nikon Eclipse Ni-U microscope.

In order to analyze the GFP distribution within the ZEs and SEs, serial optical sections of the embryos were obtained using a confocal laser scanning microscope (CLSM; system FLUO-view 1000; Olympus). The GFP was excited using a multi-Argon Laser (laser power 100 mV; Melles Griot BV; Max. 150 mW) at a 488 nm wavelength and an emission at 500–530 nm. Targeted embryos at each stage of development were studied with an objective lens at different magnifications (UPlanFLN 10x-0.30 numerical aperture, UPlanFLN 20x-0.50 numerical aperture, UPlanFLN 40x-1.35 numerical aperture). Observations were also made using an Olympus BX42 epifluorescence microscope equipped with an Olympus XC50 digital camera and software (Nikon, Tokyo, Japan). The GFP was excited at a maximum wavelength of 490 nm [Nikon Plan Fluor 10x objective lens (0.30 numerical aperture); 20x (0.5 numerical aperture); and 40x (0.75 numerical aperture)]. The histological images were acquired with a Nikon Eclipse Ni-U microscope equipped with a Nikon Digital DS-Fi1-U3 camera and software (Nikon, Tokyo, Japan).

Metacyclic trypomastigotes were obtained in vitro using chemically defined conditions as described previously^{63 (link)}. Briefly, exponential epimastigotes were washed with PBS and resuspended in TAU medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 8 mM phosphate buffer pH 6.0) to a density of 5 × 10⁸ parasites/ml with and without Tet (0.5 μg/ml) for 2 hs at 28 °C. Then, they were diluted 1:100 in TAU3AAG Medium (TAU medium plus 10 mM Glucose, 2 mM L-Aspartic Acid, 50 mM L-Glutamic Acid and 10 mM L-Proline) and incubated at 28 °C for 72 hours in the absence or presence of Tet. Parasites were collected, fixed in 4% paraformaldehyde solution and stained with Giemsa to be visualized with a Nikon Eclipse Ni-U microscope and counted using ImageJ software⁶⁴ . Only parasites with a fully elongated nucleus and rounded kinetoplast at the posterior end of the parasite were considered as metacyclic forms. Five hundred parasites from each triplicate were counted and the experiment was repeated three times.

Cells grown on the cover-slip were fixed for 20 min in 4% paraformaldehyde solution (Sigma) in phosphate-buffered saline (PBS, Euroclone). Cells were permeabilized for 30 min in blocking solution, containing 0.2% Triton X-100 (Sigma) and 10% donkey serum (Sigma) and incubated overnight at 4 °C with the primary mAb in blocking solution. The following mAbs specific for Flavivirus E protein (1:200, Millipore, MAB10216), double-stranded RNA (1:300, English and Scientific Consulting Kft, Hungary), vimentin (1:300, Bioss, BS-0756R) and calreticulin (1:300, Sigma, C4606) were used. Cells were then washed with PBS and incubated for 1 h with Hoechst and either anti-mouse Alexa Fluor-488 or anti-rabbit Alexa Fluor-594 secondary Abs (1:1,000 in blocking solution, ThermoFisher Scientific). High resolution wide field fluorescence images were acquired on a Nikon Eclipse Ni-U microscope equipped with a Nikon 60x plan apo 1.40 oil or a 20x plan apo 0.75 objective and a Nikon DS-Qi2 camera.