runx2/cbfa1 antibody  (Bio-Techne corporation)

Journal: Journal of dental research

Article Title: A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

doi: 10.1177/00220345231208175

Figure Lengend Snippet: Figure 1. Parathyroid hormone–related protein (PTHrP) is expressed in the superficial layer adjoining RUNX2+SOX9neg cells in the polymorphic layer. (A) Structure of Pthrp-mCherry knock-in allele. In Pthrp-mCherry allele, red fluorescent protein mCherry is expressed instead of a functional PTHrP protein, constituting a null reporter allele. (B, C) Sagittal section of PthrpmCherry/+ mandible at postnatal day 3 (P3). (B) Magnified view of mandibular condylar cartilage (MCC). Pthrp messenger RNA RNAscope assay (in situ hybridization [ISH]). Scale bar: 20 µm. (C) Overview of the mandible. Co.pr, condylar process; Go.pr, gonial process; IAN, inferior alveolar nerve; In, incisor; M1, first molar; M2, second molar. Red: PTHrP- mCherry (IHC), yellow: SOX9-Alexa647 (IHC), gray: DAPI. Scale bar: 500 µm. n = 4 mice. (D, E) PthrpmCherry/+ MCC at P3. (D) Scale bar: 50 µm. (d1) Magnified view of the superficial, polymorphic, and flattened chondrocytes layers. (d2) Magnified view of the hypertrophic chondrocytes layer. Red: PTHrP-mCherry (IHC), blue: RUNX2-Alexa647 (IHC), magenta: SOX9-Alexa750 (IHC), green: Col10a1-Opal520 (ISH), gray: DAPI. Scale bars: 30 µm. n = 4 mice. (E) Magnified view of MCC. Scale bar: 10 µm. (F) Hierarchical structure of MCC at P3, hematoxylin and eosin staining (left) and diagram (right) highlighting PTHrP, RUNX2, SOX9, and Col10a1 expression.

Article Snippet: After RNAscope, sections were incubated in blocking buffer (3% bovine serum albumin/Tris-buffered saline with Tween (TBST)) for 30 min at room temperature (RT), and subsequently stained with SOX9 polyclonal antibody (1:500; R&D systems, AF3075), RUNX2 polyclonal antibody (1:100; Novus Biological, NBP1-89104), PTHrP polyclonal antibody (1:100; Invitrogen, PA5-57493), PTH1R monoclonal antibody (3D1.1) (1:200; Novus Biological, NBP1-40067), DsRed Polyclonal Antibody (Living Colors) (1:700; Takara Bio, 632496), or mCherry monoclonal antibody (16D7) (1:400; Invitrogen, M11217), overnight at 4°C.

Techniques: Knock-In, Functional Assay, RNAscope, In Situ Hybridization, Staining, Expressing

Journal: Journal of dental research

Article Title: A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

doi: 10.1177/00220345231208175

Figure Lengend Snippet: Figure 3. Parathyroid hormone–related protein (PTHrP) promotes cell proliferation in the polymorphic layer and formation of SOX9+ chondrocytes. (A) Hematoxylin and eosin (H&E) staining of Control (PthrpmCherry/+) and PTHrP-KO (PthrpmCherry/mCherry) mandibular condylar cartilage (MCC) at E15.5 and E18.5. fc, flattened chondrocyte; hc, hypertrophic chondrocyte; pm, polymorphic; sf, superficial. Scale bars: 50 µm (E15.5), 100 µm (E18.5). (B) Cell proliferation assay of Control (PthrpmCherry/+) and PTHrP-KO (PthrpmCherry/mCherry) MCC at E15.5 and E18.5 by EdU. White boxes (100 × 100 µm): areas used to quantify EdU+ cells. Red: PTHrP-mCherry (in situ hybridization [ISH]), yellow: EdU-Alexa480, gray: DAPI. Scale bars: 100 µm. Right: quantification of EdU+ cells in the polymorphic layer. Blue line: Control, red line: PTHrP-KO. n = 4 mice per each group. Two-tailed, 1-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Data are presented as mean ± SD. A P value of <0.05 was considered significant (*P < 0.05: between Control and PTHrP-KO, #P < 0.05: between E15.5 and E18.5). (C) Magnified views of the polymorphic layer highlighting EdU and RUNX2. Red: PTHrP-mCherry (IHC), blue: RUNX2-Alexa647 (IHC). Scale bars: 30 µm. n = 4 mice. (D) Length of SOX9+ domains (double-headed arrows) in Control and PTHrP-KO MCC at E15.5 and E18.5. Red: PTHrP-mCherry (IHC), magenta: SOX9-Alexa647 (IHC). Scale bars: 50 µm (E15.5), 100 µm (E18.5). Right: quantification of SOX9+ domain. Blue line: Control, red line: PTHrP-KO. SOX9+ domain is significantly reduced in PTHrP-KO MCC at E18.5. n = 4 mice per each group. Two-tailed, 1-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. A P value of <0.05 was considered significant (*P < 0.05: between Control and PTHrP-KO, #P < 0.05: between E15.5 and E18.5).

Article Snippet: After RNAscope, sections were incubated in blocking buffer (3% bovine serum albumin/Tris-buffered saline with Tween (TBST)) for 30 min at room temperature (RT), and subsequently stained with SOX9 polyclonal antibody (1:500; R&D systems, AF3075), RUNX2 polyclonal antibody (1:100; Novus Biological, NBP1-89104), PTHrP polyclonal antibody (1:100; Invitrogen, PA5-57493), PTH1R monoclonal antibody (3D1.1) (1:200; Novus Biological, NBP1-40067), DsRed Polyclonal Antibody (Living Colors) (1:700; Takara Bio, 632496), or mCherry monoclonal antibody (16D7) (1:400; Invitrogen, M11217), overnight at 4°C.

Techniques: Staining, Control, Proliferation Assay, In Situ Hybridization, Two Tailed Test

Journal: Journal of dental research

Article Title: A PTHrP Gradient Drives Mandibular Condylar Chondrogenesis via Runx2.

doi: 10.1177/00220345231208175

Figure Lengend Snippet: Figure 5. Parathyroid hormone–related protein (PTHrP) preferentially promotes Runx2 expression in the polymorphic layer. (A) Runx2 and Sp7 (encoding Osterix) expression in Control (PthrpmCherry/+) and PTHrP-KO (PthrpmCherry/mCherry) mandibular condylar cartilage (MCC) polymorphic layer at E15.5 and E18.5. (a1) Runx2 expression at E15.5 and 18.5. Red: PTHrP-mCherry (in situ hybridization [ISH]), green: Runx2-Opal520 (ISH). Right: quantification of Runx2 expression level. (a2) Sp7 expression at E15.5 and E18.5. Red: PTHrP-mCherry (IHC), green: Sp7-Opal520 (ISH). Right: quantification of Sp7 expression level. n = 4 mice per each group. Two-tailed, 1-way analysis of variance followed by Tukey’s post hoc test. Data are presented as mean ± SD. A P value of <0.05 was considered significant (*P < 0.05: between Control and PTHrP-KO, #P < 0.05: between E15.5 and E18.5). (B) Concluding diagram. PTHrP released from the superficial layer acts on cells in the polymorphic layer, promotes proliferation of chondrocyte precursor cells in the polymorphic layer, and prevents their premature differentiation by stimulating Runx2 expression through the PTHrP-PTH1R signaling pathway.

Article Snippet: After RNAscope, sections were incubated in blocking buffer (3% bovine serum albumin/Tris-buffered saline with Tween (TBST)) for 30 min at room temperature (RT), and subsequently stained with SOX9 polyclonal antibody (1:500; R&D systems, AF3075), RUNX2 polyclonal antibody (1:100; Novus Biological, NBP1-89104), PTHrP polyclonal antibody (1:100; Invitrogen, PA5-57493), PTH1R monoclonal antibody (3D1.1) (1:200; Novus Biological, NBP1-40067), DsRed Polyclonal Antibody (Living Colors) (1:700; Takara Bio, 632496), or mCherry monoclonal antibody (16D7) (1:400; Invitrogen, M11217), overnight at 4°C.

Techniques: Expressing, Control, In Situ Hybridization, Two Tailed Test