Fig 1: Double immunofluorescence staining for H2B-GFP- (green) and IGFBP5 (red) (a, b, e, f) or TUNEL assays (red) (c, d, g, h), GFP-immunoreactivity (i, k), and in situ hybridization against Igfbp5 (j, l) in the autografted teeth at 3 (a-d, i, j) and 7 (e-h, k, l) days after operation. Quantitative analyses of the proportions of H2B-GFP- (m) or TUNEL-positive cells (o) to pulpal cells and IGFBP5- or TUNEL-positive cells to H2B-GFP-LRCs (n). The result of the RT-PCR analysis of the ß-actin, IGFBP3, IGFBP5, and IGF-I gene expression levels in the dental pulp cells obtained from control and autografted teeth at 3 and 7 days after operation (p) (*P values are <0.05, **P values are <0.01). Doxycycline was administered to a mother via drinking water to label pups at E15.5 to induce GFP transgene expression and 3-week-old animals were used for the operation. (o) On Day 3, the number of TUNEL-positive cells in the odontoblasts and in the subodontoblastic layer are significantly increased, although apoptotic activity is not upregulated in the center of the dental pulp. (a, b, m, n) Intense and faint H2B-GFP-LRCs remain in the center of the pulp chamber, and 76.2% of these LRCs show concomitant IGFBP5-positive reactions. (c, d, n) In contrast, only 5.3% of the LRCs in the center of the pulp chamber show a TUNEL-positive reaction. (o, h) On Day 7, the number of TUNEL-positive cells in the dental pulp are significantly decreased. (e, f, n) H2B-GFP-expressing LRCs are distributed throughout the dental pulp, and 76.8% of these LRCs show IGFBP5 expression. (k) Some, but not all, LRCs are committed into newly-differentiated odontoblast-like cells. (j, l, p) On postoperative Days 3–7, the expression levels of IGF-I, IGFBP3, and IGFBP5 in the dental pulp are confirmed by the results of the in situ hybridization and RT-PCR analysis. (D dentin, DP dental pulp, OB odontoblast, Sub OB subodontoblastic cells, CP the center of the dental pulp). Bars 100 µm (a-h), 50 µm (i-l).
Fig 2: In situ hybridization of Igfbp5 (a, e, i, m) and the IGFBP5- (b, f, j, n), IGF-I- (c, g, k, o), and IGFBP3-immunoreactivities (d, h, l, p) in developing mouse upper first molars from postnatal 1 day to 4 weeks. j, k, and l are a higher magnification of the boxed area in (f), (g), and (h), respectively. (a, b) On postnatal Day 1, an IGFBP5-positve reaction is observed in the nuclei of differentiated odontoblasts, and these cells express weak Igfbp5. IGFBP5-positive cells are scattered throughout the dental papilla. (e, f, i, j) On postnatal Week1, cells at the pulp horn intensely express IGFBP5/Igfbp5. Some enamel epithelial cells and surrounding bone cells also express IGFBP5. (m,n) On postnatal Week 4, cells in the center of dental pulp and subodontoblastic layer show intense IGFBP5-positive reaction. (c, d, g, h, k, l) The dental pulp broadly express intense IGF-I and faint IGFBP3, although differentiated odontoblasts lack these positive reactions. An IGFBP3-positive reaction is not localized in the nuclei of the pulpal cells, but rather in the extracellular matrix. (D dentin, DP dental pulp, E enamel, OB odontoblasts). Bars 250 µm (a, e), 100 µm (f-h, m), 50 µm (b-d, i, n-p), 25 µm (j-l).
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