Fig. 9.

Immunogold labeling of KCNN4 channels in apical (A), lateral (L), and basal (B) plasma membranes in cryothin sections of normal rat distal colon (RtDC) and normal human colon (HuC) of surface epithelium. Cryosemithin sections were prepared and trypan blue stained for orientation (left column of each panel). Tissue specimens were then ultratrimmed for cryothin sectioning focusing on small areas of interest in the surface epithelium to characterize the plasma membrane domain-specific localization of KCNN4 proteins on the ultrastructural level. Cryothin sections were immunolabeled with anti-KCNN4-abc and detected with a secondary donkey anti-rabbit 10-nm gold-labeled antibody. Anti-KCNN4-abc localized KCNN4-like proteins in A, and L plasma membrane domains of rat colonic epithelial cells, but not in B membranes of surface epithelial enterocytes sectioned. These high-resolution immunogold electron microscopy results are consistent with and confirm our observations made by confocal microscopy. S, surface epithelium; C, crypts; bm, basement membrane/collagen. Images were acquired either at ×400 primary magnification (cryosemithin section, bar = 20 μm) by light microscopy or at ×21,000 primary magnification (cryothin sections, bar = 200 nm) by electron microscopy. Similar results were obtained with two and three different human and rat tissues, respectively.