Progres cf camera

Transgenic sticklebacks were generated by microinjection of freshly fertilized eggs as previously described (Chan et al., 2010 (link)). Plasmids were co-injected with Tol2 transposase mRNA as described (Hosemann et al., 2004 (link)). Mature Tol2 mRNA was synthesized by in vitro transcription using the mMessage mMachine SP6 kit (Life Technologies). All enhancer assays were performed on pelvic-complete stickleback from Matadero Creek, California, USA (MATA). All larvae were raised under standard aquarium conditions to Swarup St 29/30 (Swarup, 1958 (link)), when pelvic bud development is initiated, for phenotyping. Larvae were anesthetized in 0.0003% w/v tricaine (Ethyl 3-aminobenzoate methanesulfonate, Sigma). Microscopic observation for GFP expression was conducted with a MZFLIII fluorescent microscope (Leica Microsystems, Bannockburn, IL) using GFP2 filters and a ProgResCF camera (Jenoptik AG, Jena, Germany) to distinguish GFP expression from autofluorescence in pigmented fish.

Microinjection of freshly fertilized Threespine Stickleback eggs with Tol2 transposase mRNA and GFP reporter constructs was performed as previously described (Chan et al. 2010 (link); Thompson et al. 2018 (link)). All resulting larvae were raised under standard aquarium conditions to Swarup stage 30 (Swarup 1958 ) and then euthanized in 600 mg/L tricaine, pH 7.5 for phenotyping. GFP expression was documented using a Leica MZFLIII fluorescence microscope (Leica Microsystems) fitted with a GFP2 filter and ProgResCF camera (Jenoptik AG). Only fish exhibiting bilateral green eyes (from background expression by the hsp70 minimal promoter of the expression construct, indicating extent of transgenesis) were phenotyped (Nagayoshi et al. 2008 (link)). The percomorph PelA constructs were assayed on lab-raised pelvic- and caudal-complete stickleback descended from fish collected at Rabbit Slough, Alaska, USA. The construct containing the Japanese Medaka ortholog of the Faf1 CONDEL candidate region was assayed on lab-raised pelvic- and caudal-complete Threespine Stickleback descended from fish collected at Matadero Creek, California, USA and at Little Campbell River, British Columbia, Canada. The experiment did not include blinding, but post hoc PCR confirmation of integrated plasmid identity was performed.

Architecture of biofilms grown in flow cells or in microtiter plates was assessed via confocal laser scanning microscopy (CLSM). CSLM was carried out using a Leica TCS SP5 confocal microscope. Prior to confocal microscopy, biofilms were stained using the BacLight LIVE/DEAD viability stain (Life Technologies) at a 1/1000 dilution in the growth medium. The CLSM images were processed using LAS AF software v2.4.1. Quantitative analysis of the images was performed using the COMSTAT^{96 (link)}. For brightfield visualization of biofilm architecture, the samples were viewed by transmitted light using an Olympus BX60 microscope and Olympus UPLNFLN 20× and 40× objectives. Images were captured using a ProgRes CF camera (Jenoptik, Jena, Thuringia) and processed using ProgRes CapturePro 2.7.7 software.