Computational protocol: Owenia fusiformis – a basally branching annelid suitable for studying ancestral features of annelid neural development

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Protocol publication

[…] Anatomical details of developmental stages of Owenia fusiformis Delle Chiaje, 1844, were revealed in whole animal preparations using standard immunohistochemical staining protocols and a range of well-established antisera as neural markers. Every staining was carried out using at least 25–30 specimens of each stage. Although the specificities of the employed antibodies have all been established in numerous invertebrates (details below), we cannot fully exclude that a given antiserum may bind to a related antigen in the investigated specimens. We hence refer to observed labelled profiles as exhibiting (antigen-) like immunoreactivity (−LIR). Negative controls were obtained by omitting the primary antibody in order to check for antibody specificity and yielded no fluorescence signal. For immunohistochemistry specimens were rinsed 2 x 5 min in PTW (PBS with 0.1 % Tween 20) at RT (room temperature) and transferred into 10 μg proteinase K/ml PTW for 2–3.5 min depending on the developmental stage (24hpf-3dpf = 90s; 7 dpf = 2 min; 14–21 dpf = 2,5 min; after metamorphosis = 3,5 min). After 2 short rinses in glycine (2 mg glycine/ml PTW), and 3x 5 min washes in PTW, the specimens were re-fixed using 4 % PFA in PBS containing 0.1 % Tween for 20 min at RT. Subsequently the developmental stages were rinsed 2x5 min in PTW, 2x5 min in THT (0,1 M TrisCl, 0,1 % Tween) and blocked for 1-2 h in 5 % sheep serum in THT according to the protocol of Conzelmann and Jekely (2012) []. The primary antibodies, polyclonal rabbit anti-serotonin (INCSTAR, Stillwater, USA, dilution 1:500), monoclonal mouse anti-acetylated α-tubulin (clone 6-11B-1, Sigma-Aldrich, St. Louis, USA, dilution 1:500), and polyclonal rabbit anti-FMRFamide (ImmunoStar Inc., Hudson, USA, dilution 1:1000) were applied for 24–72 h in THT containing 5 % sheep serum at 4 °C. Specimens were then rinsed 2x 10 min in 1 M NaCl in THT, 5x 30 min in THT and incubated subsequently with secondary fluorochrome conjugated antibodies (goat anti-rabbit Alexa Fluor 488, Invitrogen, USA, dilution 1:500; goat anti-mouse Alexa Fluor 633, ANASPEC, Fremont, USA, dilution 1:500) in THT containing 5 % sheep serum for 24 h at 4 °C. Subsequently, the samples were washed 6x 30 min in THT, stained with DAPI for 10–15 min (5 mg/ml stock solution, working solution: 2 μl in 1 ml THT – final concentration 10 μg/ml) and washed 2x 5 min in THT. For clsm-analyses samples were mounted on glass slides using 90 % glycerol/10 % 10x PBS containing DABCO. Specimens were analyzed with the confocal laser-scanning microscope Leica TCS STED (Leica Microsystems, Wetzlar, Germany). Confocal image stacks were processed with Leica AS AF v2.3.5 (Leica Microsystems), ImageJ and Imaris 6.3.1 (Bitplane AG, Zurich, Switzerland). The final panels were designed using Adobe (San Jose, CA, USA) Photoshop CC and Illustrator CC. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Owenia fusiformis, Drosophila melanogaster
Chemicals Serotonin