For double-staining with OCT4/MAGE-A4, the MAGE-A4 was detected with Streptavidin Al-exafluor 647 (Molecular Probes) via a biotinylated rabbit anti-mouse secondary antibody, and OCT4 was visualized by tyramide-enhanced Cy3 via an HRP-conjugated rabbit anti-goat secondary antibody.
To localize C-KIT, MAGE-A4, and CHK2 in the same section, the three primary antibodies and their respective detection systems were applied to the sections sequentially. First, C-KIT primary antibody was applied to the section and detected by a directly labeled goat anti-rabbit-Alexafluor546 secondary antibody (Molecular Probes).
This was followed by MAGE-A4 primary antibody detected as above by Streptavidin-488, this time via a biotinylated goat anti-mouse secondary antibody. Finally, anti-CHK2 antibody was applied to the section and was detected as described above for the double-staining.
Nonfluorescent images were photographed using a Provis microscope (Olympus Optical, London, U.K.) and a Kodak DCS330 digital camera (Eastman Kodak, Rochester, NY). Fluorescent images were captured using a Zeiss LSM Axiovert 100M confocal microscope (Carl Zeiss Ltd., Welwyn Garden City, U.K.). Images were compiled using Photoshop 7 (Adobe, Mountain View, CA).