Anti fmrfamide

Six specimen of Owenia fusiformis were relaxed for immuno-histochemical investigations in a 7% MgCl₂ seawater (1:1) solution and subsequently fixed in 4% paraformaldehyde in seawater. Animals were embedded into Gelatine-albumen (Sigma- Aldrich) and cut into sections of 60 μm thickness with a vibratome (HM 650 V Thermo-Scientific) or intact animals were used as whole mounts. Animals were stained with antibodies against FMRF-amide (ImmunoStar, Hudson, WI, USA), acetylated α-Tubulin (Sigma-Aldrich, Saint Louis, MO, USA) and for the nucleus staining Sytox (Invitrogen, Carlsbad, CA, USA). For a detailed description see Beckers et al. [51 (link)]. For whole mount staining the tissue of Owenia fusiformis was permeabilized using 2% Triton X-100 in PBS for 65 h in the fridge. Antibodies of rabbit anti- FMRF-amide (ImmunoStar, Hudson, WI, USA) and mouse α-tubulin (Sigma-Aldrich, Saint Louis, MO, USA) were applied for 3 days after blocking in 6% swine serum in PBS containing 0.5% Triton-X-100. Secondary antibodies were applied for 2 days at a dilution of 1: 1000. Animals were treated with Murray clear (benzyl benzoate + benzyl alcohol) and embedded therein on glass slides. Vibratome sections were embedded using Elvanol on glass slides. Afterwards the slide preparations were scanned with a Leica TCS SPE CLSM. Image stacks were further processed using Fiji (1. 52 h).

All animals were fixed in 4% PFA (in 1X PBS, 0.1% Tween20) for 30 min. Larvae 48 h and older were first relaxed with 1:1 MgCl₂-seawater for 3–5 min before fixing them in 4% PFA (in 1X PBS, 0.1% Tween20) for 30 min. After fixation, the samples were washed two times in PTW, followed by two washes in THT (0.1 M Tris pH 8.5, 0.1% Tween20). Blocking was in 5% sheep serum in THT for 1 h before incubating in primary antibodies (Monoclonal anti-acetylated α-tubulin, 1:300 Sigma product number T6793; Anti-5-HT, 1:500, Immunostar product number 20080; Anti-FMRFamide, 1:500, Immunostar product number 20091) for 48 h at 4 °C. The samples were then subjected to two 10 min washes in 1 M NaCl in THT followed by five 30 min washes in THT before incubating in secondary antibodies (Alexa Fluor 1:500, Thermo Fisher Scientific) overnight at 4 °C. Next, the samples were washed in THT, two 5 min washes followed by five 30 min washes. Specimens were stored in embedding medium (90% glycerol, 1x PBS, and 2% DABCO) at 4 °C.