Journal: Breast Cancer Research : BCR

Article Title: Osteoblasts are “educated” by crosstalk with metastatic breast cancer cells in the bone tumor microenvironment

doi: 10.1186/s13058-019-1117-0

Figure Lengend Snippet: EOs are present in patient samples of bone metastatic breast cancer. Human patient samples of bone metastatic breast cancer were stained using multi-plex immunofluorescence for RUNX2 (green), osteocalcin (OCN, red), IL-6 (purple), and alpha-SMA (yellow). Left panel—osteoblast identification: white arrows show osteoblasts positive for both RUNX2 and OCN. Middle panel—“uneducated” and “educated” osteoblast identification: blue arrows show “uneducated” osteoblasts alpha-SMA and IL-6 positive; yellow arrows show “educated” osteoblasts alpha-SMA high, but IL-6 low; purple arrows show “educated” osteoblasts IL-6 high, but alpha-SMA low. Right panel—“educated” osteoblast identification: green arrows show “educated” osteoblasts both IL-6 and alpha-SMA low, DAPI positive. T, tumor; arrows, osteoblast. DAPI, nuclear stain. Scale bar = 50 μm

Article Snippet: The slides were incubated overnight at 4 °C with either mouse anti-human osteocalcin (R&D Systems, 10 μg/ml), rabbit anti-human RUNX2 (Abcam, 1:500), mouse anti-human alpha-smooth muscle actin (1:100, Abcam), rabbit anti-human alkaline phosphatase (1:100, Abcam), rabbit anti-human osteopontin (1:500, Abcam), or goat anti-human IL-6 (40 μg/mL, R&D Systems), Next, slides were incubated for 1 h at room temperature with either donkey anti-mouse 594, chicken anti-rabbit 594, donkey anti-goat 488, or goat anti-rabbit 488 (1:1000, Biotium).

Techniques: Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Divide-and-conquer strategy with engineered ossification center organoids for rapid bone healing through developmental cell recruitment

doi: 10.1038/s41467-025-61619-y

Figure Lengend Snippet: a , b HE staining of paraffin sections in Defect, Vehicle, Hybrid, and OCO groups at 4 weeks and 8 weeks after calvarial defect (bar = 200 μm at low magnification and bar = 50 μm at high magnification). c , d Safranin-O staining of paraffin sections in Defect, Vehicle, Hybrid, and OCO group at 4 weeks and 8 weeks after calvarial defect (bar = 200 μm at low magnification and bar = 50 μm at high magnification). e Schematic of neurogenesis in the central area and f xo-immunofluorescent staining of CGRP and β-III tubulin expression (bar = 20 μm); g schematic of angiogenesis in the interlayer and h co-immunofluorescent staining of CD31 and COLX expression (bar = 20 μm); i schematic of osteogenesis in the outer layer and j co-immunofluorescent staining of OCN and OSX expression (bar = 20 μm); in Defect, Vehicle, Hybrid, and OCO groups at 4 weeks post-surgery. At least three times of experiments were repeated independently.

Article Snippet: The samples were treated with primary antibodies at 4 °C overnight, including Ki67 (1:250 dilution, ab16667, Abcam, USA), γh2AX (1:250 dilution, ab22551, Abcam, USA), RUNX2 (1:200 dilution, ab76956, Abcam, UK), OSX (1:200 dilution, ab209484, Abcam, UK), Collagen I (1:100 dilution, ab260043, Abcam, UK), OCN (1:50 dilution, MAB1419, R&D Systems, USA), NGFR (1:50 dilution, NBP2-67296, NOVUS, USA), CGRP (1:100 dilution, ab81887, Abcam, UK), beta III Tubulin (1:500 dilution, ab18207, Abcam, UK), Collagen X (1:100 dilution, 14-9771-82, Invitrogen, USA), CD31 (1:100 dilution, ab222783, Abcam, UK), CD200 (1:100 dilution, AF2724, R&D Systems, USA), Thy1 (1:200 dilution, ab181469, Abcam, UK), KRT8 (1:100 dilution, ab53280, Abcam, UK), HAS1 (1:250 dilution, PA5-95599, Invitrogen, USA), MSX1 (1:100 dilution, ab93287, Abcam, UK).

Techniques: Staining, Expressing

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: A novel mouse model to study fracture healing of the proximal femur.

doi: 10.1002/jor.24677

Figure Lengend Snippet: FIGURE 4 Immunohistochemical staining of fractured femurs at day 14 (d14) and day 21 (d21) after fracture. A, Representative images of fractured femurs stained for collagen X and quantification of the positively stained area in the whole fracture callus. B, Representative images of fractured femurs stained for runx2 and quantification of the positively stained area in the whole fracture callus. C, Representative images of fractured femurs stained for osteocalcin and quantification of the positively stained area in the whole fracture callus. Scale bar = 200 µm. C, cortex; G, fracture gap. *.05 > P > .01; ***.01 > P > .0001 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Paraffin‐embedded 7‐μm longitudinal sections were prepared for immunohistochemical staining, the following antibodies were used: rabbit anti‐mouse collagen X (ABIN1077945, dilution 1:200; Antibodies Online), rabbit anti‐mouse Runx2 (8486, 1:50; Cell Signaling), rabbit anti‐mouse osteocalcin (orb77248, 1:200; Biorbyt), goat‐anti rabbit IgG‐biotin (sc‐3840, 1:200, Santa Cruz) and horseradish peroxidase‐conjugated streptavidin (Zytomed Systems).

Techniques: Immunohistochemical staining, Staining

Journal: Cell Transplantation

Article Title: Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing

doi: 10.3727/096368912x654920

Figure Lengend Snippet: Figure 3. Osteogenic differentiation of cEx-BM CD34+ cells and BM CD34+ cells in vitro. (A–D) Morphology of cEx-BM CD34+ and BM CD34+ cells, which were cultured in α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 10% conditioned medium (CM) during the first 7 days and in the same medium without CM for the following 1 week, characterized by a fibroblast-like spindle shape (original magnification: 200×) (A, B). These spindle-shaped cells prolifer- ated quickly forming colonies (original magnification: 40×) (C, D). (E, F) In wells with osteogenesis-inducing conditions, matrix mineralization was clearly demonstrated by Alizarin Red staining, indicating the presence of calcium. In contrast, no mineralization was observed under noninducing conditions (original magnification: 40×). Each upper right panel shows the entire image of the well. (G) Quantification of Alizarin Red staining. The Alizarin Red-positive area (red) and total area in the field were measured, and the percentage of Alizarin Red-positive area to total area was calculated and averaged in cEx-BM CD34+ cells versus BM CD34+ cells. NS, not significant. (H) mRNAs of human osteocalcin (hOC) and human collagen 1 α1 (hCol1A1) were markedly expressed in osteogenesis-induced cells (Os+) but not in noninduced cells (NI). (I–K) Immunofluorescence staining of human osteocalcin-positive cells (original magnification: 40×). Scale bars: 100 μm. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: To detect differentiation of transplanted human cells in the rat perifracture sites and to evaluate capillary density and osteoblast (OB) density, immunohistochemistry was performed with the following human- or rat-specific antibodies: human nuclear antigen (hNA) (1:50 dilution, Chemicon, Billerica, MA, USA) as a sensitive marker for all transplanted human cells, biotinylated Ulex europaeus agglutinin 1 (UEA-1) (1:100 dilution; Vector Labora tories, Bur lingame, CA, USA) as a sensitive marker for human endothelial cells (hECs), fluorescein-labeled isolectin B4 (FITC-conjugated ILB4) (1:100; Vector Laboratories) as a sensitive marker for rat ECs (rEC), anti-human osteocalcin (hOC) antibody (1:50; Biomedical Technologies, Inc., Stoughton, MA, USA) as a marker for human OBs, and anti-rat osteocalcin (v-19) (rOC) (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as a marker for rat OBs (rOB).

Techniques: In Vitro, Cell Culture, Staining, Immunofluorescence

Journal: Cell Transplantation

Article Title: Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing

doi: 10.3727/096368912x654920

Figure Lengend Snippet: Figure 7. Enhancement of angiogenesis and osteogenesis by recipient cells following cEx-BM CD34+ and BMCD34+ cell transplan- tation. (A) Closed square in toluidine blue staining (×2) is the region of interest (ROI) observed by fluorescence vascular staining. Dotted square is the ROI observed by fluorescence osteoblast staining. Black dotted, curved line marks the edge of the callus forma- tion. (B) Angiogenesis assessed by capillary density at week 2 was significantly greater in the cEx-BMCD34 group compared to the other groups (n = 5 in each group). *p < 0.05, **p < 0.001. (C) OB density was significantly greater in the cEx-BMCD34 and BM CD34+ (Hi) groups than in the other groups (n = 5 in each group). **p < 0.001. (D) Representative vascular staining with isolectin B4 (green) and DAPI (blue) using tissue samples at perifracture sites collected at week 2 in all groups (original magnification: 100×). (E) Representative osteoblast staining with anti-rat osteocalcin (arrows, red) and DAPI (blue) using tissue samples at perifracture sites collected at week 2 in all groups (original magnification: 100×). (F–I) Gene expression of intrinsic cytokines for angiogenesis and osteogenesis at week 2 (n = 4 in each group). Relative expression levels of rat-specific vascular endothelial growth factor (VEGF) (F), angiopoietin-1 (Ang-1) (G), bone morphogenetic protein-2 (BMP-2) (H), and BMP-4 (I) to rGAPDH. The expression level of rVEGF and rAng-1 was significantly greater in animals of the cEx-BMCD34 and BMCD34 (Hi) groups compared with the other groups (F, G). The expression levels of rBMP-2 and rBMP-4 were also significantly greater in animals of the cEx-BMCD34 and BMCD34 groups compared with the BMCD34 (Lo) and PBS groups (H, I). *p < 0.05, **p < 0.001.

Article Snippet: To detect differentiation of transplanted human cells in the rat perifracture sites and to evaluate capillary density and osteoblast (OB) density, immunohistochemistry was performed with the following human- or rat-specific antibodies: human nuclear antigen (hNA) (1:50 dilution, Chemicon, Billerica, MA, USA) as a sensitive marker for all transplanted human cells, biotinylated Ulex europaeus agglutinin 1 (UEA-1) (1:100 dilution; Vector Labora tories, Bur lingame, CA, USA) as a sensitive marker for human endothelial cells (hECs), fluorescein-labeled isolectin B4 (FITC-conjugated ILB4) (1:100; Vector Laboratories) as a sensitive marker for rat ECs (rEC), anti-human osteocalcin (hOC) antibody (1:50; Biomedical Technologies, Inc., Stoughton, MA, USA) as a marker for human OBs, and anti-rat osteocalcin (v-19) (rOC) (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as a marker for rat OBs (rOB).

Techniques: Staining, Fluorescence, Gene Expression, Expressing