Fig. 5. Immunocytochemistry and immuno-electron microscopy of AH7974 cells. AH7974 cells were stained with anti-rnSMase 1 antibody and the localization of enzyme was observed by immuno-fluorescence microscopy (A-G) and immuno-electron microscopy (H,I) as described in Materials and Methods. (A) Phase contrast of AH7974 cells. (B) Cells in A stained for nSMase 1 with anti-rnSMase 1 antibody (green fluorescence of FITC). (C) Cells in A stained for cytoplasm with anti-KDEL antibody (red fluorescence of Cy^TM3). (D) Cells in A stained of nuclear DNA with DAPI. (E) Phase contrast of AH7974 cells. (F) Control staining of cells as in E with the antibody pre-treated with purified rat nSMase 1 at 4°C for 3 hours. (G) Merge of two pictures of confocal laser microscopy stained with anti-rnSMase 1 (green) and anti-KDEL (red). (H) Immuno-electron microscopy of an AH7974 cell with anti-rnSMase 1 antibody. Localization of the enzyme was shown by gold colloid particles (10 nm) conjugated with second antibody. (I) Control staining of immuno-electron microscopy without first antibody. Bar, 200 nm. Abbreviations: Nuc, nucleus; Cyt, cytoplasm; arrows indicate clusters of gold particles, and open arrowheads indicate nuclear envelope.