Leica application suite las x

We examined the morphology of 17 dried and pinned specimens belonging to P.ivani (nine specimens) and P.caraganella (eight specimens). The adults of both species were photographed with Leica digital microscope DMS1000 and the incorporated digital camera and processed using the stacking system software Leica Application Suite LAS X. Genitalia were dissected from five P.ivani and four P.caraganella moths (Suppl. material 2: Table S2) and their photographs were taken with Sony Nex3 Camera from Carl Zeiss Stemi DV4 Stereo Microscope. Leaf mines were photographed in the field and in the laboratory using a digital camera Sony Nex3. All images were edited in Adobe Photoshop CS5 Extended.
Genitalia dissection and slide mounting followed Robinson (1976) . Terminology of the genitalia followed Klots (1970) and Kristensen (2003) (link).

Rhodamine 123 transport assays were performed in the VBDOC with only cholangiocytes in the device. The 5 μm Rhodamine 123 (Thermo Fisher Scientific) in CCCM was perfused to the basal side through the endothelial channel (without endothelial cells), resulting in it diffusing through the whole chamber, and normal CCCM was added to the cholangiocyte channel. The cholangiocyte channel ends were then blocked with vacuum grease to ensure that there was no Rhodamine 123 in the lumen at the start of the assay and to avoid Rhodamine 123 dilution by medium in the reservoir ports. After incubating for 2 h at 37 °C, the basal side was washed 3× with PBS via the endothelial channel.
In the inhibition assay, cholangiocytes were incubated with 10 μm Verapamil in CCCM at 37 °C for 30 min, followed by incubation with Rhodamine 123, as described above. Images were acquired using a Leica confocal microscope and Leica application suite (LAS X; Leica, Buffalo Grove, IL, USA). Fluorescence intensity was measured across the cholangiocyte channel.

At 48 h, engineered islets were fixed with 4% v/v paraformaldehyde for 15 min at room temperature and rinsed three times in phosphate buffered saline (PBS, Sigma-Aldrich, UK). Nonspecific sites were then blocked with 5% w/v bovine serum albumin (BSA, Sigma-Aldrich, UK) containing 0.2% v/v Triton X in PBS for 1 h and stained with a primary antibody against CD31 (1:50, R&D system, UK), PDX-1 (1:10, Abcam, UK), insulin (1:50, Cell Signalling technology, UK) and Connexin 43 (CX43, 1:50, R&D system, UK). All primary antibodies were diluted in a 0.5% w/v BSA in PBS and incubated overnight at 4 °C. Following washings, islets were incubated in goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 and goat anti-rabbit IgG secondary antibody conjugated Alexa Fluor 555 (1:100, Fisher-Scientific, UK) for 1 h at room temperature, rinsed twice with PBS and counterstained with Hoechst 33342 (1 ug/mL, Cell Signalling technology, UK) for 5 min. Images were finally captured using both fluorescence microscopy with digital SLR camera (Nikon Eclipse TE2000-U) and a confocal scanning microscopy (Leica TCS-SP5).
Z-stack images of each 3D cell constructs were also taken and processed using Leica software (Leica Application Suite, LAS X).