Fig. 1

Distribution of rhIGF-1/rhIGFBP-3 in the immature brain of the preterm rabbit pup. A. Outline of experimental in vivo setup. B and C. Representative three-dimensional light-sheet microscopy images illustrating the presence of AF647-labeled rhIGF-1/rhIGFBP-3 (in white) in the ChP, perivascular and subarachnoid space. D. Representative two-dimensional light-sheet microscopy images illustrating distinct presence of rhIGF-1/rhIGFBP-3 in the ChP of the lateral and third ventricle (white, lower), and to a lesser degree in the fourth ventricle (white, upper). Light-sheet images were collected from 4 independent experiments. E-J. Representative confocal microscopy images illustrating biotin-labeled rhIGF-1/rhIGFBP-3 (subsequently visualized with streptavidin-coupled AF647) in ChP of the lateral ventricle (E, red), the SFO in the third ventricle (F-G, red), and negligible presence in the parenchyma (G-H, red). I-J. Visualization of CD31 at the endothelial lining of the SFO (I, green) and co-existence with rhIGF-1/rhIGFBP-3 (J, CD31 green, rhIGF-1/rhIGFBP-3 red). Cell nuclei were counterstained with DAPI (E-J, blue). Confocal images were collected from 3 independent experiments. K-L. Representative TEM images illustrating rhIGF-1/rhIGFBP-3 labeled with FITC (subsequently visualized with anti-FITC gold conjugate) at the basal (K) and the apical (L) border of the ChP in the lateral ventricles. White and black arrows display gold-labeled rhIGF-1/rhIGFBP-3. TEM images were collected from 2 independent experiments. C.S., cesarean section; GA, gestational age; E, embryonic; P, postnatal; LV, lateral ventricle; SFO, subfornical organ; 3V, third ventricle. For additional experimental details, see Methods and Results section