For the gastrozooid-expressed JFT, HsymJFT1c-I, polyclonal antibodies were produced by AbClonal using their Rabbit Polyclonal Antibody Development For Protein service. For the dactylozooid-specific JFT, HsymJFT1c-II, polyclonal antibody was designed, expressed, and purified from GenScript Biotech using their PolyExpress Premium Antigen-Specific Affinity Purified pAb service. Goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor 594 (Invitrogen, Cat. A-11012) was used as the secondary antibody. Anti-human Histone H1 (Leinco Technologies Inc. Prod. # H126) and Goat anti-mouse IgG (H+L), F(ab%)2 fragment, CF 488A antibody Secondary Alexa (Sigma-Aldrich, Cat. SAB4600388) were used as controls. Immunostaining was carried out as in Sanders et al. [41 (link)]. The DAPI staining occurred in 9:1 glycerol/PBS media, which reduces the staining in capsules of maturing and mature nematocysts. Epifluorescence images were taken using a Leica DM5000 B with attached Lumenera Infinity 3s and Infinity Analyze v7 software. Confocal images were taken either using a Leica Laser Scanning Confocal Upright Microscope at the University of Kansas Microscopy and Analytical Imaging Core or LeicaTCS SP5 with LAS AF software. Images were processed using Fiji (ImageJ2) v2.9.0/1.53t [46 (link)] and all final figures were designed using Inkscape [40 ]. An alignment of all studied JFTs in this work shows antigentic regions selected for antibody production in Supplementary Figure S5 [47 (link),48 (link)]. A lack of HsymJFT1c-II staining in gastrozooids is shown in Supplementary Figure S6 .
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