To localize the expression of ANO1-like and SCN5A-like ion channels in Hydra using immunocytochemistry, polyclonal antibodies were raised against synthetic peptides in rabbits. The peptides that correspond to intracellular loops located between transmembrane domains of the ion channels (hySCN5-like: SRSKPKMFKDYKPE; hyANO-like: ETRRPIADRAQD) were synthetized, purified, and N terminally conjugated with KLH prior to injection (GenScript). Polyclonal antibodies were affinity-purified on the antigen and concentrated to 1.5 mg/mL. Serum harvested from the rabbits prior to their immunization was used as control.
Immunohistochemical detection in whole-mount Hydra preparations was carried oud as described previously (77 (link)). Briefly, polyps were relaxed in urethane, fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking solution for 1 h, and incubated further with primary antibodies diluted to 1.0 μg/mL in blocking solution at 4 °C. AlexaFluor488-conjugated goat anti-rabbit antibodies (Invitrogen) were diluted to 4 μg/mL and incubations were done for 1 h at room temperature. Rhodamin-phalloidin (Sigma) and TO-PRO3-iodide-AlexFluor633 (Invitrogen) counterstaining was conducted as described previously (77 (link)). Confocal laser-scanning microscopy was done using a TCS SP1 laser scanning confocal microscope (Leica).
Immunohistochemical detection in whole-mount Hydra preparations was carried oud as described previously (77 (link)). Briefly, polyps were relaxed in urethane, fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, incubated in blocking solution for 1 h, and incubated further with primary antibodies diluted to 1.0 μg/mL in blocking solution at 4 °C. AlexaFluor488-conjugated goat anti-rabbit antibodies (Invitrogen) were diluted to 4 μg/mL and incubations were done for 1 h at room temperature. Rhodamin-phalloidin (Sigma) and TO-PRO3-iodide-AlexFluor633 (Invitrogen) counterstaining was conducted as described previously (77 (link)). Confocal laser-scanning microscopy was done using a TCS SP1 laser scanning confocal microscope (Leica).
