Ultrastructural detection of alpha1-fetoprotein in hepatomas by use of peroxidase-labelled antibodies
Wolf D. Kuhlmann
Immunocytochemistry SFB 136 and Institut für Nuklearmedizin, DKFZ Heidelberg, Germany
Hepatomas were induced in rats by feeding N-nitrosomorpholine. Nodules from alpha-1-fetoprotein (AFP) producing hepatoma were taken and subcutaneously injected into syngeneic hosts. From the first generation, the hepatoma producing most AFP was selected for further transfers. Four weeks after transplantation, the AFP concentration in the host rats was in the range of 0.8 to 1.5 mg AFP/ml serum. Transplanted hepatomas of the 14th to 19th generations were used for ultrastructural localization of AFP. Various methods of tissue processing were examined under the light and electron microscopes for intracellular detection of AFP by use of specific anti-AFP antibodies conjugated with peroxidase. AFP was localized in the perinuclear space, the rough-surfaced endoplasmic reticulum and the Golgi apparatus. Non-membrane-bound ribosomes did not stain for AFP. Fixation with 6% formaldehyde for 5 hours followed by 6% formaldehyde plus 0.25-0.5% glutaraldehyde for 60-90 minutes proved best both for satisfactory ultrastructural conservation of the organs and for immunocytochemical localization of AFP. Concentrations of glutaraldehyde which exceeded 0.5% led to a significant decrease in immunocytochemical AFP reactions. On the other hand, weak aldehyde fixation poorly preserved ultrastructural detail and leakage of insufficiently fixed material caused staining artifacts. Under our conditions, thick frozen sections from well-fixed hepatomas gave consistently reproducible results in that intracellular penetration of antibody-peroxidase conjugates was superior to hand-cut small tissue fragments. Tissue fixation and sampling were most important above all for other experimental steps.