Freeze-fracture replica electron microscopy combined with sds digestion for cytochemical labeling of integral membrane proteins: Application to the immunogold labeling of intercellular junctional complexes

Abstract

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cyto-chemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.