Figure 9.

Localisation of RBP4 and GPC3 in suture tissue. A-B) Immunofluorescence and H&E stain showing intense localisation (yellow) of RBP4 in the cytoplasm of osteocytes (oc) in ectocranial surface bone (unfused coronal suture, #83). C-D) Serial immunofluorescence (C) and H&E sections (D) showing RBP4 located in cells in the region between calcified tissue (b) and mesenchyme (m) (unfused left lambdoid suture, #83). E) RBP4 was not detected on the endocranial surface of unfused sutures (coronal, #83). F) RBP4 was localised to the cytoplasm of osteoblasts (ob) lining the developing bone, those being trapped in the osteoid (arrow head), and osteocytes (unfused coronal suture, #83). G) Corresponding phase contrast image to the central region in (F). H) RBP4 was not detected in fused sutures. Red blood cells had weak autofluorescence (sagittal, #5). I-L) GPC3 immunofluorescence (I) and H&E (J) detected protein in mesenchymal cells close to the tissue surface (arrow head) in the mid-suture region (unfused sagittal suture, #5). Membrane staining was observed for the cytoplasmic extensions of mesenchymal cells adjacent to calcified bone (K-L, unfused coronal suture, #83). J) H&E of section deep to (K) showing calcified bone protruding into intervening mesenchyme with osteoblasts lining the bone. Scale: 10 μm