Axiocam 503 colour camera

Suture tissue specimens harvested from patients, were fixed in 4% (w/v) formalin in phosphate buffer 0.1 M at pH 7.2 for 72 h at 4 °C. After rinsing with running water, the samples were decalcified in Decalcifier DC2 solution (#11028306 VWR Chemical, Radnor, PA, USA) for 24–96 h (based on sample size) at 4 °C under constant agitation. The tissues were then washed in phosphate buffer saline (PBS) 0.01 M pH 7.4 for 1–2 h at RT, and then cryoprotected in 15% sucrose in PBS overnight at 4 °C. The samples were then embedded in OCT compound, and 10-µm-thick sections were cut on a cryostat (CM 1850, Leica, Wetzlar, Germany) and directly mounted on slides. The slides were used both for haematoxylin and eosin staining and for immunofluorescence analysis. In particular, for histological analysis, the sections were incubated with haematoxylin (according to Mayer, 05-06002/L Bio-Optica, Milan, Italy) for 5 min, washed in running water for 5 min, and then stained with eosin (05-10002/L Bio-Optica, Milan, Italy) for 1 min. Finally, the sections were dehydrated through a series of graded alcohols, clarified in xylene and cover-slipped with Eukitt mounting medium (03989 Sigma Aldrich, Saint Louis, MO, USA). The slides were then examined using a Zeiss Axiophot microscope and the images were captured with an AxioCam 503 colour camera (Zeiss, Jena, Germany) coupled to the microscope.

Standard Drosophila husbandry procedures were followed. Stocks were maintained on a potato-mash based diet at room temperature. Crosses were performed at 25°C and 60% humidity level. The UAS-ALK-L1198F and UAS-ALK-G1201E constructs were generated using standard cloning techniques and transgenic flies were obtained by injection (BestGene Inc.). white¹¹¹⁸ (w¹¹¹⁸) flies were used as control; GMR-Gal4 (#1104) was received from the Bloomington Drosophila Stock Center at Indiana University (BDSC; NIH P40OD018537). UAS-ALK, UAS-ALK-F1174L, UAS-ALK-I1250T as well as UAS-FAM150A (AUG-β) and UAS-FAM150B (AUG-α) were used for ectopic expression studies in the developing Drosophila eye employing the GAL4-UAS system. Staining of imaginal discs was described previously [38 (link)]. The rabbit monoclonal ALK (D5F3) antibody was purchased from Cell Signalling and employed at a 1:200 dilution. The hybridoma, monoclonal mouse antibody 4F3 (anti-discs large; employed at 1:500), deposited by Corey Goodman was obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the NICHD of the NIH and maintained at The University of Iowa. Samples were analysed under Zeiss Axio Imager.Z2 and AxioZoom.V16 microscopes. Images were acquired with a Zeiss LSM800 confocal microscope or with an Axiocam 503 colour camera employing ZEN blue edition software.

Z-stack photographs were acquired with a Zeiss Axio Zoom.V16 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a PlanApo Z 1.0×/0.25 FWD 60 objective and an AxioCam 503 colour camera. Images were processed with the software Zen 2 and Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, USA) by cropping, contrast enhancement and removal of the background.
Ecological photographs were taken with EF 100 mm f/2.8L IS USM and MP-E 65 mm f/2.8 1-5X lenses attached to a Canon 5D Mark IV SLR camera. Images and plates were processed on a standard Windows 10 platform using Adobe Photoshop CS6 (Adobe Systems, Inc., San Jose, CA, USA).
Measurements and terminology follow Hastriter and Bush (2006) (link).