Fig. 8. Immunofluorescence analysis of COS7 cells transfected with envoplakin C-terminal constructs, alone (A-H) or in combination with the periplakin P-1/4R+L construct (I-L). Cells were fixed without prior extraction (A-C,E-G,I) or extracted before fixation (D,H,J-L). Green fluorescence: anti-FLAG (to detect tagged envoplakin). Red fluorescence: anti-vimentin (A,C,D,E,G,H); anti-HA (to detect tagged periplakin; I,K,L). Blue/purple fluorescence: nuclei stained with TOTO-3. Boxed inserts in A,E,I are shown at higher magnification in A-C, E-G and I, respectively. J-L are the same field. In D there is no residual green fluorescence and therefore no evidence that the cells shown were actually transfected. In H the central cell has weak residual green fluorescence. Bars, 10 µm.