Localization of glycolytic enzymes in murine erythrocytes resealed in the presence or absence of an antibody to the cytoplasmic domain of band 3. Freshly drawn murine erythrocytes were washed 3 times to remove serum and buffy coat, and divided into 2 aliquots. One suspension of erythrocytes was lysed and resealed in the presence of rabbit polyclonal antibody raised against cdb3.1 The other suspension (control) were similarly lysed and resealed in the absence of any antibody. After resealing, both erythrocyte preparations were similarly fixed with acrolein, permeabilized with Triton X-100 (“Preparation of cells”), and stained with the desired antibodies. (i) Antirabbit antibody conjugated to Cy2 (492 510) was used to identify cells containing entrapped anti-cdb3 antibody. (ii) Cells (lacking entrapped antibody) were first stained with the same rabbit anti-cdb3 antibody and then labeled with Cy2-conjugated antirabbit IgG. Membrane staining in this panel demonstrates the specificity of the antibody for band 3. (iii,iv) Stained first with goat antibodies specific for either aldolase (A) or pyruvate kinase (B) and then with antigoat antibody conjugated to Cy5 (650 670). (v,vi) Overlays of subpanels i and iii, and ii and iv, respectively. (vii,viii) Bright field images of subpanels i, iii, v, and ii, iv, vi, respectively.
