Alexa fluor 488 conjugated goat anti rabbit immunoglobulin

Immunofluorescence assays (IFAs) were performed on air-dried smears fixed with 4% paraformaldehyde (PFA) for 10 min, followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min, and blocked with 3% BSA in PBS blocking buffer overnight at 4°C. Incubation with primary antibodies was carried out for 1 h followed by secondary-antibody incubation for 45 min. The primary antibody used was PfGAP45 (rabbit, 1:5,000). The secondary antibody used was Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin (1:1,000) from Invitrogen. After incubation with primary and secondary antibodies, nuclei were stained with Hoechst dye for 10 min, and the slides were mounted with VectaShield Vibrance medium. The primary antibodies α-tubulin (mouse) and GAP45 (rabbit) were supplied by J.D. The secondary antibodies used were Alexa Fluor 594-conjugated goat anti-mouse immunoglobulin (1:1,000) and Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin (1:1,000) from Invitrogen. After incubation with primary and secondary antibodies, nuclei were stained with Hoechst dye for 10 min, and the slides were mounted with anti-fade Vectashield medium. The preparations were visualized under a Zeiss LSM 880 microscope with Airyscan 2 for high-resolution confocal images.

Antibody labelling was performed as previously described [8 (link)]. Briefly, tube feet were fixed in 4% PFA in phosphate buffered saline (PBS) for 24 h. They were subsequently dehydrated in graded ethanol, embedded in paraffin wax and cut longitudinally. After dewaxing and rehydration, antigen retrieval with a solution of 0.05% trypsin and 0.1% CaCl₂ was performed for 15 min on 37 °C. Footprints were collected on microscope glass slides and fixed in 4% PFA in PBS overnight at room temperature. All samples were blocked in PBS containing 3% (w/v) bovine serum albumin (BSA) for 30 min at room temperature. Antibodies directed against Sfp1α and Sfp1β were diluted 1:100 in blocking solution and added to samples for 2 h at room temperature. Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulins (Invitrogen) were applied 1:100 diluted in blocking solution for 1 h at room temperature. In the negative controls, no primary antibody was added and only the secondary antibody was applied. Samples were analysed with a Zeiss Axioscope A1 microscope.

The immunohistochemistry on tube foot sections was performed with some alterations to a previously published protocol [11 (link)]. Tube feet were fixed in 4% (w/v) paraformaldehyde (PAF) in phosphate-buffered saline (PBS; pH7.4), rinsed in PBS, and subsequently dehydrated in a graded ethanol series. Tube feet were then embedded in paraffin wax and cut longitudinally into 5 µm-thick sections with a Microm HM 340 E microtome. After dewaxing and rehydration, antigen retrieval was achieved by incubation in a solution containing 0.05% (w/v) trypsin (Sigma) and 0.1% (w/v) CaCl₂ for 15 min at 37°C. Samples were blocked in PBS containing 3% (w/v) bovine serum albumin (BSA) and 0.3% (v/v) Triton X-100 for 30 min at room temperature. The polyclonal anti-Asterias rubens Sfp7/8, Sfp10 and Astacin-like Sfp antibodies were diluted 1 : 100 in blocking solution and added to samples for 1 h at room temperature (see antibody production and electronic supplementary material, S2). Alexa Fluor 488-conjugated goat-anti-rabbit immunoglobulins (Invitrogen) were diluted 1 : 200 in blocking solution and applied for 1 h at room temperature. Samples were mounted in Vectashield mounting medium (Vector Laboratories) and analysed with an Olympus FV1000 confocal microscope.