Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules

Abstract

were processed for immunocytochemistry. Immunogold labeling using antibodies against the N-termlnus (Glyl-Hisl3), Leu siSGin s28 and the C-terminus (Ilel°°2-Tyr t°°6) was superimposed on 2. Materials and methods the images of the electron microscope protoplasmic fracture face of the basolateral plasma membranes, thus demonstrating Tissue preparation and quick:[reezingThe inner stripe of the outer medulla of rat kidneys was cut into cytoplasmic locations of these epitopes. On the contrary, SDSsections less than 100 ~m thick by a Microslicer (Dosaka EM Co.,

Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas

“…In his experiments, SDS digestion was used to dissolve unfractured cell components before the immunocytochemical reactions that permitted the labeling of antigens in freeze-fractured intracellular membranes. The SDS-digested freeze-fracture replica labeling technique (SDS-FRL) has been applied to studies of epitope topology of intramembranous protein molecules (Fujimoto et al 1996a) and transmembrane topology of membrane phospholipids (Fujimoto et al 1996b).…”

A simple and reliable quick-freezing/freeze-fracturing procedure

“…We previously reported that integral membrane proteins revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody (Fujimoto 1995;Furuse et al 1996). In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes (Fujimoto et al 1996b) and the precise topological analysis of integral membrane proteins using site-directed antibodies (Fujimoto et al 1996a). In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon (Pt/C) shadowing, are treated with 2.5% SDS.…”