Fig 4.

Immunofluorescence staining using Abs against peptides derived from Sto1p, Sto4p, and Sto8p. Cells were fixed and permeabilized to achieve optimal conditions for the immunoreaction as indicated in Materials and Methods. Cells then were labeled by affinity-purified Abs against peptides specific for the different Stomatin families, recognizing Sto1a,b,cp (A), Sto4a,b,c (B), and Sto8a,bp (C, red), respectively, as explained in the text. Counterstaining was performed with a mouse monoclonal anti-α-tubulin Ab (clone DM1A; green; Sigma) and with the secondary Abs specified in Materials and Methods. Note the patchy arrangement of Stomatin at the cell surface outside cilia (column a) with stronger staining in the region of the oral cavity (column b), as well as the occurrence of some Stomatin in granular form inside cells (column c). The contractile vacuole complex (column c) as well as the porus (column d) are stained only by Abs against Sto1a,b,cp and Sto4a,b,cp, whereas anti-Sto4a,b,cp stains some only phagocytic vacuoles (column e). fv, food vacuole. Scale bars, 10 μm.