Immunofluorescence and Immunohistochemistry Analysis of EO and HEO Cell Complexes

Cell complexes of EO and HEO were fixed in a 4% (w/v) solution of paraformaldehyde. For immunohistochemistry, the cell complexes were embedded in paraffin and sliced according to standard protocols. The paraffin sections then underwent standard procedures for immunohistochemical staining.

For immunofluorescence, the fixed cell complexes were permeabilized with a 0.1% solution of Triton in PBS for 1 day. The incubation period was extended to 2 days for primary antibodies and to 1 day for secondary antibodies. The nuclei were stained with DAPI overnight. After each incubation step, the samples were washed with PBS three times for 30 minutes per wash.

Immunofluorescence images were obtained using a confocal laser-scanning microscope (ZEISS 800, ZEISS, Oberkochen, Germany). Movie files of 3D rendered images were created using Zeiss ZEN Black 2010 software (Carl Zeiss Canada).