Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: Sheep MSC were characterized using FACS and RT-PCR analysis. (A) With RT-PCR analysis CD29, CD44 and CD166 expression of MSC could be proofed on mRNA level. As indicated by increased CD45 expression, ratio of hematopoietic cells was higher in directly auto-transplanted MSC as compared to expanded MSC. (B–D) FACS analysis revealed sheep MSC to express CD29, CD44 and CD166. Expanded MSC (B) were negative for the hematopoietic markers CD31 and CD45. Directly auto-transplanted cells (C) had a different expression pattern than expanded MSC. The directly auto-transplanted MSC had a weaker CD29 and CD166 but a stronger CD45 expression. Mean fluorescent indices are shown in (D).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were DiI labelled prior to implantation and implanted subcutaneously with or without BMP-2. (A–C) Expanded MSC (A), directly auto-transplanted MSC (B), BMP-2 in combination with directly auto-transplanted MSC (C). DiI-labelled MSC (red) could be found close to β-TCP/HA granules contributing to the newly formed bone parts. In the explants with directly auto-transplanted MSC a higher section of the DiI-labelled cells were found in the connective tissue parts of the constructs compared to the explants with expanded MSC or directly auto-transplanted MSC with BMP-2. (D–F) Sections of constructs of the groups with expanded MSC (D), directly auto-transplanted MSC (E), BMP-2 in combination with directly auto-transplanted MSC (F) were evaluated for vascularization. The constructs in all three groups are well vascularized as shown by CD31 immunohistochemistry (green). Nuclei are counterstained with DAPI (blue).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Construct, Immunohistochemistry

Journal: Oncotarget

Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

doi: 10.18632/oncotarget.10474

Figure Lengend Snippet: A. DBA-1 mice were injected with parental or CREB-deficient HER-2/neu + cells as described in Materials and Methods and tumors were removed after 42 days. Representative photos of parental and CREB-deficient HER-2/neu + tumors are shown. The arrows indicate the blood vessels on the tumor surface. The tumor volume is given. The bar represents 1 cm (left). 5 μm slices of paraffin-embedded tumors were stained with the indicated primary antibody followed by an anti-rabbit secondary antibody. The detection was performed with the peroxidase substrate DAB. Slides were counterstained with methylene blue. The arrow heads indicate the blood vessels. The bar represents 100 μm; Magnification: 40x (right). B. The blood vessel density of the tumors was analysed by counting vessel structures in the anti-CD31 mAb-stained samples (see 1A). Bars represent mean values from four samples/group with four counted fields/sample. C. The necrotic area was analysed in the HE-stained samples. Bars represent mean values from four samples/group with four counted fields/sample. D. The hypoxic area was analysed in the anti-HIF-1α-stained samples. Bars represent mean values from four samples/group with four counted fields/sample. E. 1×10 4 HUVEC/well were seeded in a 96 well plate on polymerized growth factor reduced matrigel. 100 μl/well fresh medium or cell conditioned medium was added and the cells were incubated for 16 h by 37°C. The morphology of the HUVEC under these distinct culture conditions was compared (left) and the mesh-like structures were quantified (right) as described by Zhang . The bar represents 80 μm; Magnification: 10x. F. 1×10 5 parental and CREB-deficient HER-2/neu + cells resuspended in matrigel were injected into the flank of female DBA-1 mice (n = 8). 7 days after injection the mice were killed and the removed matrigel plugs were photographed. The bar represents 1 cm (up). 5 μm slices of the matrigel plugs from parental and CREB-deficient HER-2/neu + cells were stained as indicated. The bar represents 100 μm; Magnification: 10x (down). G. Matrigel plugs from parental and CREB-deficient HER-2/neu + cells were homogenized and their hemoglobin content was analysed as described as in Material and Methods. The bars represent the hemoglobin concentration of each plug from mice injected with the indicated cell line normalized to the weight of the plug. Data demonstrate the results of one out of two independent experiments (with five Matrigel plugs in each experiments) regarding the hemoglobin content/plug from parental and CREB-deficient HER-2/neu + cells.

Article Snippet: Tumor samples were cut into 5 μm slices and then incubated with primary mAb directed against CD31 (Abbiotec) or HIF-1α (Novus) for 24 h. A compatible secondary antibody linked with HRP SignalStain ® Boost IHC Detection Reagent (Cell Signaling) was used for the detection with the substrate DAB.

Techniques: Injection, Staining, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

doi: 10.18632/oncotarget.10474

Figure Lengend Snippet: Primary and labeled secondary antibodies used in this study

Article Snippet: Tumor samples were cut into 5 μm slices and then incubated with primary mAb directed against CD31 (Abbiotec) or HIF-1α (Novus) for 24 h. A compatible secondary antibody linked with HRP SignalStain ® Boost IHC Detection Reagent (Cell Signaling) was used for the detection with the substrate DAB.

Techniques: Labeling, Western Blot, Staining, Flow Cytometry, Ubiquitin Proteomics