Detection of antigens by immunofluorescence on ultrathin cryosections of skin

Cryoultramicrotomy was originally established to provide ultrathin cryosect ions as substrates for on-section immunolabeling in immunoelectron microsco py. Recently, we recognized that ultrathin cryosections of skin (0.2 mu m t hick) could serve as substrates for immunofluorescence (IF) with excellent resolution. To assess the advantages and the limitations of IF on ultrathin cryosections we compared the labeling of IF on 0.2-mu m ultrathin cryosect ions of skin with those of routine IF on 6-mu m cryostat sections, confocal laser scanning microscopy (LSM), and immunogold electron microscopy using several markers of keratinocyte cell surface and basement membrane zone mol ecules. IF on ultrathin cryosections clearly demonstrated a lack of bullous pemphigoid antigens beneath the melanocytes, desmosomal antigens as discon tinuous dot-like labeling, and nondesmosomal plasma membrane antigen as a l adder-like pattern. IF on ultrathin cryosections provided convincing images with higher resolution than confocal LSM, which corresponded well to those of immunogold electron microscopy. IF on ultrathin cryosections had superi or resolution compared to routine IF or confocal LSM and should serve as a powerful tool in future studies for the analysis of skin antigens.