Figure 3
Immunofluorescence detection of MCP-1 and heparan sulfate by confocal microscopy; the green channel shows FITC-stained heparan sulfate and the red channel represents TRITC-stained MCP-1. (a) Representative paired X–Y images showing relatively uniform distribution of heparan sulfate (green) but punctate distribution of MCP-1 (red) on the endothelial cell surface following incubation with MCP-1 in the subendothelial compartment. (b,c) Two-color X–Z sectional images showing characteristic distributions of MCP-1 associated with single cells (b) and concentrated into discrete regions of the apical cell surface (c) following incubation with subendothelial MCP-1; yellow regions indicate colocalization of heparan sulfate and MCP–1. (d) Immunofluorescence staining after apical application of 12.5 nM MCP-1 for 60 min showing focal distribution of chemokine on the apical cell surface, which is similar to that produced by basal chemokine application. (e) Immunodetection of the P8A monomeric mutant MCP-1 following basal application at 12.5 nM; again, the staining shows a focal distribution on the apical cell surface.
