Ti2 e a1rhd25

Footprints were either obtained by turning individual sea stars on their backs and letting them attach strongly to a glass slide placed on their tube feet, or by allowing sea stars to walk over a glass slide. In both cases, the sea stars and glass slides were submerged in seawater. Footprints were fixed in 4% (w/v) PAF in PBS solution, rinsed in PBS solution, dehydrated in ethanol and stored in 70% ethanol. Upon use, they were rehydrated in distilled water followed by Tris-buffered saline (25 mmol l⁻¹ Tris, 125 mmol l⁻¹ NaCl, pH8.0) containing 0.05% (v/v) Tween-20 (TBS-T) and submitted to the immunolabelling method detailed earlier, without the antigen retrieval step. Double immune-labelling was performed with the polyclonal anti-Asterias rubens Sfp1β [6 (link)] and the lectin WGA. This antibody was diluted 1 : 100 and the lectin WGA was diluted at a concentration of 25 µg ml⁻¹ in TBS-T containing 3% (w/v) bovine serum albumin (TBS-T-BSA) [6 (link),13 (link)]. Alexa Fluor 488-conjugated goat-anti-rabbit immunoglobulins (Invitrogen) were diluted 1 : 200 and Texas-Red-conjugated streptavidin (Vector Laboratories) was diluted 1 : 100 in TBS-T-BSA. Footprints were observed by using an Olympus FV1000 or Nikon TI2-E-A1RHD25 a confocal microscope.