Axioimager z1 apotome 213 system

T. castaneum dissections were carried out in Ringer’s saline and the different appendages were mounted directly in Glycerol 70%. For D. melanogaster immunohistochemistry, wings from 0 h after puparium formation animals were collected and stained as described [33 (link)]. Antibodies: mouse anti-Broad-Complex (1:100; Developmental Studies Hybridoma Bank (DSHB)); rat anti-Spalt (Sal) (1:200; a gift from R. Barrio); rabbit anti-Caspase 3 (1:250; Cell Signaling Technologies); Alexa Fluor 555-conjugated secondary antibodies (1:200; Molecular Probes). All samples were examined with AxioImager.Z1 (ApoTome 213 System, Zeiss) microscope, and images were subsequently processed using Adobe photoshop.

All ovaries were dissected in PBS, fixed with 3.7% formaldehyde/PBS and incubated with Phalloidin. The stained ovaries were mounted in Vectashiled with DAPI for epifluorescence microscopy. Dissected pupal appendages, were fixed with 3.7% formaldehyde/PBS and mounted in Vectashield with DAPI to visualized the nucleus. All pictures were obtained with AxioImager.Z1 (ApoTome 213 System, Zeiss) microscope, and images were subsequently processed using Fuji and Adobe photoshop.

Images were obtained with the Leica TCS SP5 and the Zeiss LSM880 and LSM780 confocal microscopes. The same imaging acquisition parameters were used for all the comparative analyses. Images were processed with the Imaris Software (Oxford Instruments), Fiji, or Photoshop CS4 (Adobe). For DNA quantification and nuclear size of salivary glands, DNA staining intensity in the salivary glands and tracheal cells was obtained from z stacked images every 0.25 μm of DAPI-stained L3 larvae. Image analysis was performed using Fiji. For the volumetric calculation of the wing pouch region in Imaris Software (Oxford Instruments), the regions of interest were selected based on the RFP fluorescence (induced by nub-Gal4) in confocal stacks that included the whole disc. The surface function of Imaris was used to segment the wing pouch and the surface volume was calculated by the software. Adult flies, nymphal parts, and adult cockroach images were acquired using AxioImager.Z1 (ApoTome 213 System, Zeiss) microscope, and images were subsequently processed using Photoshop CS4 (Adobe).