Fig. 9. Deposition of laminin 5 is restricted to the rear of the cell and colocalizes with sites of collagen removal. (A-C) Removal of collagen colocalized with sites of laminin 5 deposition. Migrating keratinocyte on Rhodamine-conjugated human placental type-I collagen (A), stained by immuofluorescence with mAb D2-1 for precursor laminin 5 (B). Images overlayed in C. (D-I) Deconvoluted optical sections at the substratum interface of migrating cells on collagen. mAb D2-1 was added for 15 minutes prior to cross-linking for 10 minutes with 0.4 mM BS3. The cells were then extracted with 1% Triton X-100 for 10 minutes, fixed with formaldehyde and processed for immunofluorescent staining for laminin 5 (D2-1; D,G), integrin 3 (E) and 2 (H). The nucleus is visible in E,H as a gray area. F,I merged images of D,E and G,H, respectively. Scale bar, 10 µm.