Ix81 inverted confocal microscope

ASI::neuropeptide::gfp/mCherry transgenes were constructed by PCR fusion, as described above. To co-localize NLP-9 and RAB-3, two different ASI promoters were used; gpa-4::nlp-9::gfp and srg-47::mCherry::rab-3 and co-injected into nlp-9 null animals. A similar approach was used to co-localize NLP-9 and NLP-14 using srg-47::nlp-9::mCherry and gpa-4::nlp-14::gfp. Animals were immobilized in 20 mM sodium azide, mounted on 3% agarose pads and then imaged for GFP and mCHERRY using sequential scanning. All microscopy was performed on an Olympus IX81 inverted confocal microscope.

alx1;alx3/+ incross progeny were fixed at 2 dpf in 4% paraformaldehyde (PFA)/PBS overnight. Their trunk/tail portions were reserved for genotyping and the heads were exposed to 1% KOH/6% H₂O₂ to remove pigmentation and washed with PBST-X (1X PBS with 0.5% TritonX-100), followed by a 5-min proteinase-K (1:5000) treatment and 30-min 4% PFA/PBS post-fix at room temperature. Embryos were blocked with PBSTD-X (1X PBST, 1% DMSO, 10% BSA, 10% goat serum) for 2 h, at room temperature, incubated with mouse-anti-zn5 primary antibody (Zebrafish International Resource Center A28175, 1:100) and then Alexa-488-conjugated goat anti-mouse secondary antibody (Invitrogen AB_10013770, 1:500). Nuclei were labeled with DAPI (Molecular Probes; 1:5000). For imaging, embryos were mounted in VectaShield (Vector laboratories) and imaged on an Olympus IX81 inverted confocal microscope with the Fluoview 1000 confocal package, using a 60x water immersion objective (NA 1.10).

Mitochondrial activity was studied using the fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide), which acts as a marker of mitochondrial activity and allows changes in the electrochemical potential of inner mitochondrial membrane to be detected. Dissected ovaries (1mm long, less than 0.5 mm thick) were rinsed in PBS and immersed in a solution with the JC-1 (1mg/ml DMSO) (Sigma) in PBS (10μl JC-1 in 2 ml PBS for 60 minutes). Both monomeric (excitation at 488nm, emission 500–550nm) as well as aggregation (excitation 488 nm, emission at 575–620nm) were registered using an Olympus FV1000 confocal system combined with an Olympus IX81 inverted confocal microscope with a 60x PlanApo water objective. The images were analyzed using the ImageJ program. Image segmentation was performed using the Multi-Otsu threshold plug-in for the ImageJ program. In order to perform the image segmentation the histogram of each image was first extended to saturate the gray scale. Then, the images were filtered using a median filter (r = 0.5). Afterwards 12-bit images were converted to an 8-bit gray scale. An algorithm then attributed pixels to the appropriate class of luminosity (255 steps of luminosity) which was arbitrarily divided into: class 1: 1 to 50; class 2: 51 to 100; class 3: 101 to 150; class 4: (the brightest): 151 to 255.

The labellum and prestomal teeth of M. domestica, Sc. Stercoraria, St. calcitrans, and Sa. Bullata were used to assess the extent of sclerotization and the possible presence of the elastomeric protein resilin. The labellum was removed with a razorblade and placed into a droplet of glycerol on a glass slide, which was then manipulated with insect pins to expose the prestomal teeth. A high-precision cover slip (12 mm diameter, thickness no. 1.5 H, Neuvitro Corporation, USA) was placed on top. The autofluorescence of mouthpart structures was studied with an Olympus IX81 inverted confocal microscope and the CY3 (531–540 nm excitation, 593–640 nm emission), DAPI (377–360 nm excitation, 447–460 nm emission), and GFP (469–535 nm excitation, 525–639 nm emission) filters. Mouthparts were imaged with autoexposure settings and a 15-slice Z-stack in Olympus cellSens software. Structures that appeared red indicated a high level of sclerotization, green indicated weak levels of sclerotization, and structures that had blue autofluorescence indicated the presence of resilin [27 (link)].