Visualizing HRP in HeLa Cells by Electron Microscopy

HeLa cells expressing HRP-KDEL-myc were fixed on coverslips with 1.3% glutaraldehyde in 0.1 M cacodylate buffer, washed in 0.1 M ammonium phosphate [pH 7.4] buffer for 1 h, and HRP was visualized with 0.5 mg/ml DAB and 0.005% H2O2 in 0.1 M ammonium phosphate [pH 7.4] buffer. Development of HRP (DAB dark reaction product) took between 5 to 20 min and was stopped by extensive washes with cold water. Cells were post-fixed in 2% OsO4+1% K3Fe(CN)6 in 0.1 M cacodylate buffer at 4°C for 1 h, washed in cold water and then contrasted in 0.5% uranyl acetate for 2 h at 4°C, dehydrated in an ethanol series and embedded in epon as for conventional EM. Ultrathin sections were counterstained with 2% uranyl acetate and observed under a FEI Tecnai 12 microscope, 4,800X magnification, equipped with a CCD (SiS 1kx1k keenView) camera and iTEM acquisition software.