Abstract
Fundamental studies were performed on the detection of IgM in the mouse spleen lymphocyte with the view of application of immunoperoxidase technique to clinical cytology. As for sampling of cells on the glass slide, especially in imprint smear, the important factor is uniform scattering of cytological specimen on the slide, excluding tissue fluid which may contain any kinds of antigen.
For this purpose, we washed out completely the cytological specimen with 1% BSA in PBS in order to remove the liquid component containing extracellular antigen (wash-out method), wet fixation was superior to dry one in two points; clear findings of positive reaction and well-preserved cellular features with minimum swelling and deformation of cells. Sufficient fixation did not reduce immunoreactivity, but gave satisfactory cytological preparation.
Best results were obtained by fixation of the wet smear with methanol-ether (1:1) for 30 minutes, and the dry smear with aceton for 30-60 minutes at 4°C. The attempt of digestion by proteolytic enzymes (pronase P and trypsin) enhanced immunoreactivity, without significant reduction of background staining. In general enzymatic digestion is to be avoided, in dry preparation, since it brings about more or less cellular degeneration, even in for short time incubation and with low concentration of the enzyme. Application of surface active agent such as tween 20 and 80 could not reduce background reaction, but increased specific immuno-reaction and enhanced endogenous peroxidase activity of the smeared cells. Among the immunohistochemical procedures, indirect method seemed to be most appropriate, because of easier technique and less background staining, so long as and-serum titers are high enough.
Simple PAP method showed almost the same stainability as the indirect peroxidase labelled technique applied to enzyme-digested specimens, although the former resulted in slightly increased background reaction.
The most favorable results were given, when the PAP method was applied to washed-out preparation above-mentioned.
These results could contribute to further application of immunoperoxidase technique to clinical cytology.
