Immunostaining was carried out by placing thin sections on nickel grids, oxidizing them with sodium metaperiodate to restore specific labeling, rinsing and floating them on drops of 1% BSA/PBS to block non-specific staining. The grids were then incubated on drops of the primary antisera, either anti-Ci-PAP-A22

or anti-Ci-MAM-A24. After washing, the sections were exposed to protein A-conjugated colloidal gold particles of either 10 or 5 nm diameter (Sigma Chemical Co, St. Louis, Missouri, USA). Finally, sections were counterstained with uranyl acetate prior to examination in the electron microscope. As a negative control the first antibody was omitted or an irrelevant one (Anti Bcl-xL, H5 mouse IgG1, Santa Cruz Biotechnology, Santa Cruz, CA, USA, no. 8392) was used. As for the production of antisera against Ci-MAM-A and Ci-PAP-A the synthetic peptides Epigenetics inhibitor were

coupled to keyhole limpet hemocyanin (KLH) and these conjugates were used as antigens to immunize rabbits [25] and [26], antisera were preincubated with KLH prior to their use to exclude the possibility that the staining was due to anti-KLH antibodies with cross-reactivity to C. intestinalis hemocyanin-like proteins. Negatives were scanned on an Epson Perfection 2480 Photo scanner and acquired MLN8237 chemical structure as TIFF files at 800 ppi and 300 ppi. All TIFF files were resampled at 300 ppi and subsequently re-sized and optimized for brightness and contrast by using Photoshop (Adobe Systems). By performing

immunoelectron microscopy with the specific antibodies against Ci-PAP-A22 and Ci-MAM-A24 on fixed samples from the naïve Ciona body and the oral siphon, the natural peptides were localized to the tunic tissue ( Fig. 1). Among the different cell types that are dispersed within the entire tunic ( Fig.1A), the Ci-PAP-A and Ci-MAM-A positive cells belong to the granulocyte population of “tunic large granule cells” and “tunic morula cells”, previously described by De Leo [30] on the basis of their morphology, and “tunic compartment cells”. The Niclosamide word “tunic” is included to emphasize that these cells are permanently resident in the tunic and to avoid confusion with the names applied to the hemocytes of the hemolymph. Tunic large granule cells are characterized by possessing a single, large compartment occupied by homogeneous fibrogranular content. The large inclusion inside the unique granule is surrounded by a thin peripheral rim of cytoplasm which contains a small nucleus, some vesicles and free ribosomes. The large granules immunoreacted with anti-Ci-MAM-A (Fig. 1B and E) and anti-Ci-PAP-A (data not shown). Particularly abundant in some areas of the oral siphon are tunic morula cells and tunic compartment cells (Fig. 1C).