Quantification of low numbers of immunogold label on Lowicryl K4M sections

Postembedding immunogold staining was used to quantify low numbers of intracytoplasmic cytokine interferon molecules in cells embedded in Lowicryl K4M by the rapid technique of Altman et al. (1984). When labelling with colloidal gold-protein A (CGPA) was performed as suggested by the manufacturers, the relatively high background precluded the statistically significant distinction of small amounts of specific label. The standard procedure of postembedding staining was modified by introducing: (1) UV-irradiation of the sections before immuno-staining; (2) 10- to 50-fold greater dilution of CGPA than recommended by the manufacturers, and (3) washing the sections at 60 degrees C between staining steps. The resulting decrease in nonspecific binding of CGPA facilitated the quantification of as few as 6-10 CGPA particles per 1,000 micron2 of the surface of the Lowicryl K4M sections at greater than a 95% confidence level.