Fig. 5. Differential staining of CEACAM1 by polyclonal and monoclonal antibodies, and effects of hyperosmotic treatment. Confluent, polarized CEACAM1-L- or CEACAM1-S-expressing MDCK cells were treated with or without hyperosmotic sucrose, fixed with 3% paraformaldehyde and immunostained with or without methanol permeabilization. The cells were stained either with the polyclonal antibody CC16 and Alexa546-conjugated secondary antibodies or with the monoclonal antibody 5.4 and Alexa488-conjugated secondary antibodies. For each sample both a z-reconstruction (a'-s') and a composite xy-field (a-s) is shown. The composite xy-fields were constructed by simultaneously showing all xy focal planes of a visual field superimposed in a single, combined xy-field. Lateral staining for CEACAM1-L was seen with CC16 both with and without sucrose treatment (b,b',d,d'), although the staining was weaker after sucrose treatment. MAb 5.4 detected lateral staining of CEACAM1-L only after sucrose treatment (h,h'). Note that mAb 5.4 hardly stained CEACAM1-S at all, whether the cells were treated with hyperosmotic sucrose or not.