Digital sight ds ri1 camera

Zebrafish larvae were collected and fixed overnight at 4 °C in phosphate buffered saline 1× solution (PBS) with 4% PFA (paraformaldehyde) as previously described (Garcia-Reyero et al., 2014 (link)). For detailed morphological observation and larvae measurements, fixed larvae were then washed several times with PBS and gradually transferred to 90% glycerol in PBS for long-term preservation and positioning under the stereomicroscope.
All individuals were observed and photographed under a Nikon SMZ1500 (NIKON Instruments INC. New York, United States ) dissecting microscope fitted with Nikon Digital Sight DSRi1 camera and NIS Elements AR software (version 3.0) (NIKON Instruments INC. New York, United States ) and saved as high resolution (3840 × 3005 pixels) tagged image file format (TIFF). All images were taken on the left side of each larva. The total body length (anterior-most part of the snout to posterior-most point of the tail, mm) and trunk thickness (mm) were obtained using a measuring tool from the NIS Elements software.

Transverse myocardial sections of the left ventricle were stained by Masson’s trichrome stain for visualization of fibrotic tissue as previously described^{56 (link)}. Light microscopy and image capturing was performed using a Nikon Eclipse Ti-S microscope and Nikon Digital Sight DS-Ri1 Camera (Nikon, Melville, NY, USA). For quantification of fibrotic tissue, pictures from each heart section (thickness of 10 μm) were taken systematically to ensure that the entire extent of the tissue section had been covered. A total of 20 heart sections was inspected (5 hearts/group). The micrographs were analysed using the ImageJ software (NIH). The percentage of fibrotic tissue in each micrograph was measured and averaged per heart. The average value for each heart was then utilized for statistical analysis.

Alcian Blue and Alizarin Red staining of intact skeletons were performed as previously described (Smits et al., 2004 (link), 2001 (link)). In short, embryos or newborn pups were humanely sacrificed and placed in hot tap water for 10 min. The skin was carefully removed using forceps and the animals were eviscerated. Fixation was carried out in 95% ethanol for 3 days, after which the bodies were stained in Alcian Blue staining solution [0.015% Alcian Blue 8GX (Sigma), 20% acetic acid, 76% ethanol] for 24 h. The bodies were subsequently washed three times with 95% ethanol (24 h for each wash). Bodies were cleared in 1% KOH for 24 h and stained for 12 h in Alizarin Red staining solution [0.005% Alizarin Red (Sigma), 2% KOH]. Bodies were then cleared in 2% KOH for 24 h. The clearing process was completed using the following ratios of 2% KOH to glycerol (80:20, 60:40, 40:60 and 20:80). Each clearing step was performed for 24 h. Pictures of the stained skeletons were taken using NIS-element AR 4.20.01 software (Nikon), using a Nikon SMZ18 stereomicroscope (10× objective, 0.75× lens) equipped with a Nikon digital sight DS-Ri1 camera.

In situ hybridization in zebrafish whole-mount embryos and heart cryosections was performed, as previously described [51 (link)]. PCR fragments of abcc6a and cmlc2 amplified from zebrafish cDNA were subcloned into pGEM-T vector (Promega). Embryos and heart cryosections were imaged on a Leica M205 FA dissecting scope equipped with Nikon Digital Sight DS-Ri1 camera (Nikon,Tokyo, Japan).

For histology, tissues were dissected, fixed in formalin and processed for paraffin embedding. Sections were stained by hematoxylin and eosin or with Vonkossa stain. We determined the expression of EGFP in the seminiferous tubule of pCX-Egfp and Amh-IRES2-Egfp transgenic mice by immunohistochemistry. Testis sections of 5 µm were subjected to immunostaining with mouse anti-EGFP (Clontech, Mountain View, CA) as primary antibody at a dilution of 1:200 and then antimouse IgG tagged to Alexafluor 488 (Molecular probes, Eugene, OR) was used at a dilution of 1:250, as secondary antibody. The fluorescence was detected by Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Chiyoda-ku,Tokyo, Japan). The images were captured using Nikon-digital sight DS-Ri1 camera.

For histology and post-mortem tissues, samples were fixed in 10% Neutral buffered formalin (NBF), paraffin embedded, sectioned at 4 μm and stained with hematoxylin and eosin. For immunohistochemistry, samples were prepared using standard methods. In brief, tissue sections were processed for staining by microwaving in 0.01M citrate buffer, pH 6. After incubation with primary antibodies (Cleaved caspase 3, Cell Signaling, #9664; γH2AX, Millipore #AB5535), samples were incubated with biotinylated secondary antibody (Vector) followed by incubation with Avidin Biotin Complex (Vector); slides were developed in 3,3′-diaminobenzidine (DAB) substrate (Vector) and counterstained in hematoxylin. Tumors and lymphomas images were taken using a Nikon Digital Sight DS-Ri1 camera paired to a Nikon 90i Eclipse microscope. Imaging software was NIS-Elements AR Ver 4.0, 64bit.