After cell fixation with PFM, the cells were washed with PBS pH 7.5 (Sigma) and permeablized with 0.1% Triton X-100 (Sigma) in Phosphate Buffered Saline (PBS) for 20 minutes. When using Mab 4A1 to identify LAMP-1 positive compartments, the cells were permeablized with 0.5% Saponin (in place of Triton X-100) for 15 minutes and 0.1% Saponin was included in all subsequent steps. Non-specific binding sites were blocked with block buffer (100 ml Tris-buffered saline supplemented with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10% Normal Goat Serum (Harlan Lab Sera) and 1% Teleostean Gelatin [Sigma]) for 0.5 h. The cells were washed with PBS and incubated with primary antibodies in block buffer for 1 h, washed again with PBS, and incubated with the appropriate species specific, Alexa-Fluor conjugated secondary antibody (also in block buffer) for 1 h. The cells were washed and the nuclei labelled with 49, 69-Diamidino-2-Phenylindole (DAPI) (Sigma) in H 2 O for 10 minutes and then washed with H 2 O. The coverslips were mounted in Vectashield mounting medium for fluorescence (Vector Laboratories) and sealed with clear nail varnish.