anti sdf 1a antibody  (Bio-Techne corporation)

Journal: Gut

Article Title: The chemokine receptor CXCR4 is expressed in pancreatic intraepithelial neoplasia

doi: 10.1136/gut.2007.143941

Figure Lengend Snippet: The chemokine CXCL12 is expressed in murine and human pancreatic intraepithelial neoplasia. Murine and human pancreata were examined for CXCL12 expression via immunohistochemistry (IHC). CXCL12 expression was present in all pancreatic intraepithelial neoplasia (PanIN) grades but there was no statistical difference in expression patterns between low grade and high grade PanIN. Panels designate varying degrees of PanIN. (A) mPanIN 1A (single asterisk) and mPanIN 1B (double asterisk). (B) Double staining of CXCR4/CXCL12 in a mouse PanIN 1B with CXCL12 in brown and CXCR4 in green, demonstrates concurrent expression of ligand and receptor, suggesting the possibility of an autocrine PanIN activation. (C), hPanIN 1B, and (D) hPanIN 2.

Article Snippet: The sections were incubated at 4°C with a polyclonal anti-CXCR4 antibody (Abcam, Cambridge, Massachusetts, USA) at 1:50 dilution for 60 min and with a polyclonal anti-SDF-1a (CXCL12) antibody (US Biological, Swampscott, Massachusetts, USA) at 1:200 dilution for 30 min. Then, the sections were labelled with a secondary antibody (Envision+; Dako, Carpinteria, California, USA), developed with diaminobenzidine (DAB), counterstained in 50% Mayer’s haematoxylin and examined at ×200 and ×400 magnifications.

Techniques: Expressing, Immunohistochemistry, Double Staining, Activation Assay

Journal: Gut

Article Title: The chemokine receptor CXCR4 is expressed in pancreatic intraepithelial neoplasia

doi: 10.1136/gut.2007.143941

Figure Lengend Snippet: Stimulation of the chemokine receptor CXCR4 leads to increased proliferation of pancreatic intraepithelial neoplasias (PanIN) in vitro which is abrogated by concominant MEK1/2 inhibition. (A) Alamar blue proliferation assays were performed in PanIN cells with increasing concentration of the CXCR4 ligand, CXCL12. Fluorescence measurements were performed at baseline as well as 12, 24, 48 and 72 h post-CXCL12 administration. PanIN cells demonstrated a dose-dependent increase in proliferation with CXCL12 administration compared to control and was statistically significant for all concentrations at all time points (p<0.05) with a 47% difference in fluorescence at 72 h between PanIN stimulated with 250 ng/ml CXCL12 and untreated control. (B) Pre- and concomitant incubation of the PanIN cells with the CXCR4 specific inhibitor AMD3100 abrogated the increased proliferation seen with CXCL12 administration alone. (C) Further decrease in proliferation when PanIN cells were co-incubated with the MEK1/2 inhibitor U0126. (D) Incubation with AMD3100 and U0126 was similar to U0126 alone, indicating that CXCL12 mediated PanIN proliferation is mitogen-activated protein kinase (MAPK) dependent.

Article Snippet: The sections were incubated at 4°C with a polyclonal anti-CXCR4 antibody (Abcam, Cambridge, Massachusetts, USA) at 1:50 dilution for 60 min and with a polyclonal anti-SDF-1a (CXCL12) antibody (US Biological, Swampscott, Massachusetts, USA) at 1:200 dilution for 30 min. Then, the sections were labelled with a secondary antibody (Envision+; Dako, Carpinteria, California, USA), developed with diaminobenzidine (DAB), counterstained in 50% Mayer’s haematoxylin and examined at ×200 and ×400 magnifications.

Techniques: In Vitro, Inhibition, Concentration Assay, Fluorescence, Control, Incubation

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: COMSOL parameters.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Concentration Assay, Diffusion-based Assay

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Spatial trends of total SDF-1a (black) immunostaining and CXCR4 (pink) cell density 1, 3, and 7 days post injection. Day 1 and day 3 bolus (a, c) spatial distribution of SDF-1a and CXCR4 declines steadily across the tissue. Day 1 and day 3 NP (b, d) spatial distribution reveals sustained levels of SDF-1a and CXCR4 across 1600 μm of tissue, suggesting production of SDF-1a must be occurring endogenously. Spatial levels diminish on day 7 for the bolus group (e) and remain persistent immediate to the injection for the NP group (f). Mean ± SEM. n = 4–5 animals per group. Single asterisks above error bars indicate significance compared to all groups within data set where *p < 0.05, **p < 0.01.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Immunostaining, Injection

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Spatial distribution of total SDF-1a 1 day after exogenous delivery. Representative cortical tissue sections from bolus delivered AFSDF-1a (top) and AFSDF-1a loaded NPs (bottom) stained for cell nuclei (DAPI, blue) and total SDF-1a (red), with EGFP-CXCR4 (green) and exogenous AFSDF-1a (pink). Increased levels of total SDF-1a immunostaining and CXCR4 are present visually across the cortex for the NP group. Immunostaining reveals steady levels of total SDF-1a across the cortex for the NP group in contrast to the spatially decreasing pattern for the bolus group. Consistent spatial levels of total SDF-1a suggest autocrine/paracrine signaling is occurring. Figure is adapted from Dutta et al. with copyright permissions.7 Scale bar = 200 μm.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Staining, Immunostaining

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Microglia (a), brain endothelial cells (b), and astrocytes (c) engage autocrine/paracrine signaling via dynamic response to exogenous SDF-1a. Significant changes in SDF-1a (black) and/or CXCR4 (pink) mRNA expression were found across all cell types. Changes in expression are dynamic and distinct over time for each cell type. n = 3–6 cultures per cell type. Mean ± SEM. *p < 0.05.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Expressing

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Diffusion only model predicts undetectable SDF-1a after 1 hour. Diffusion-only model concentration plots of soluble SDF-1a for bolus (a) and NP (b) delivered SDF-1a across 1 hour, 1 day, 3 days, and 7 days. Arc length (X-axis) represents equidistant space from the injection. Separate colors/line styles used to distinguish between time points (1 hour, 1 day, 3 days, 7 days). Lack of concentration plot lines indicates undetectable soluble SDF-1a present at corresponding time points. Both bolus (a) and NP (b) trends are not consistent with in vivo data, suggesting another mechanism is required to sustain SDF-1a signal spatiotemporally.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Diffusion-based Assay, Concentration Assay, Injection, In Vivo

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Autocrine/paracrine model replicates in vivo results. Autocrine/paracrine model concentration plots of soluble SDF-1a for bolus (a, c) and NP (b, d) delivered SDF-1a across 1 hour (black), 1 day (pink), 3 days (teal), and 7 days (purple). Arc length (X-axis) represents equidistant space from the injection. Addition of autocrine/paracrine kinetics flattens concentration curves of both SDF-1a and CXCR4, yielding spatiotemporal sustainment and matching most in vivo results.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: In Vivo, Concentration Assay, Injection

Journal: Cellular and Molecular Bioengineering

Article Title: Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain

doi: 10.1007/s12195-020-00643-y

Figure Lengend Snippet: Temporal trends of total SDF-1a immunostaining and CXCR4 cell density 1, 3, and 7 days post injection. Total SDF-1a (black) and CXCR4 (pink) immunopositivity were persistent across 7 days for the NP group (b) and gradually declined over 7 days for the bolus group (a). Overall sustainment of total SDF-1a immunopositivity and CXCR4 activity across the 7 day time window suggests autocrine/paracrine signaling. Mean ± SEM. n = 4–5 animals per group. *p < 0.05, **p < 0.01.

Article Snippet: 25 μ m sections were blocked, permeabilized, and incubated with rabbit polyclonal anti-SDF-1a (Abcam, Cambridge, MA) overnight at 4°C followed by a 2 h incubation with goat anti-Rabbit IgG Alexa Fluor555 (Thermo Fisher Scientific, Waltham, MA) at room temperature.

Techniques: Immunostaining, Injection, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells

doi: 10.3389/fcell.2020.580131

Figure Lengend Snippet: Hypoxia enhances the expression of the HIF-1a and the expression of its downstream pro-angiogenic genes in ASCs. (A) Hypoxia caused an O 2 -concentration-dependent increase in HIF-1a protein levels after 24 h ( n = 5, * denotes P < 0.05 vs. the 21% O 2 group, # denotes P < 0.05 vs. the 2% O 2 group). (B,C) The degree and duration of hypoxia regulate mRNA expression of the pro-angiogenic genes (VEGF-A, VEGFR2, HGF, SDF-1a, CXCR4, and bFGF) in ASC ( n = 6, * denotes P < 0.05 vs. the 21% O 2 group; # denotes P < 0.05 vs. the 2% O 2 group). (D) Normoxic preconditioning (NPC) indicates treatment of ASCs with 21% O 2 for 24 h; hypoxic preconditioning (HPC) indicates treatment of ASCs with 2% O 2 for 24 h. (E,F) Representative western blots of VEGFR2 and CXCR4 in ASCs with HPC and NPC. Quantification of VEGFR2 and CXCR4 protein levels (normalized to β-actin protein levels) was expressed as relative ratios ( n = 5, * denotes P < 0.05 vs. NPC group). (G,H) HPC increases VEGF-A and SDF-1a secretion in the conditioned medium from ASCs (ASC CM ) compared to the NPC ( n = 10, *denotes P < 0.05 vs. NPC-ASC CM ).

Article Snippet: To clarify the contribution of SDF-1a and VEGF-A in HPC-ASC CM -induced angiogenesis, the HPC-ASC CM was incubated with neutralizing antibodies against VEGF-A (R&D, 25 μg/mL) and/or SDF-1a (Abcam, 10 μg/mL) and was continuously stirred at 4 °C for 2 h to allow the resin to bind to the neutralizing antibodies.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells

doi: 10.3389/fcell.2020.580131

Figure Lengend Snippet: Wound healing assay in vitro . (A) The representative images of the HUVECs from each group at 0 and 24 h after scratching with a pipette head. Scale bar = 100 μm. (B) Quantitative analysis on wound closure as measured by the width covered by the migrated cells ( n = 5, *denotes P < 0.05 vs. NPC-ASC CM ; # denotes P < 0.05 vs. HPC-ASC CM + Anti-VEGF-A/SDF-1a).

Article Snippet: To clarify the contribution of SDF-1a and VEGF-A in HPC-ASC CM -induced angiogenesis, the HPC-ASC CM was incubated with neutralizing antibodies against VEGF-A (R&D, 25 μg/mL) and/or SDF-1a (Abcam, 10 μg/mL) and was continuously stirred at 4 °C for 2 h to allow the resin to bind to the neutralizing antibodies.

Techniques: Wound Healing Assay, In Vitro, Transferring

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells

doi: 10.3389/fcell.2020.580131

Figure Lengend Snippet: The tube formation assay in vitro . (A) Images of human umbilical vein endothelial cells after 24 h incubation with conditioned medium from ASC were analyzed by using the Image J plugin “Angiogenesis analyze” for Image J software. Scale bar = 200 μm. (B) The number of tubes, junctions, branches and total tube length were measured in each group. Image analysis was performed on three random fields per sample ( n = 3 samples, *denotes P < 0.05 vs. NPC-ASC CM ; # denotes P < 0.05 vs. HPC-ASC CM + anti-VEGF-A/SDF-1a).

Article Snippet: To clarify the contribution of SDF-1a and VEGF-A in HPC-ASC CM -induced angiogenesis, the HPC-ASC CM was incubated with neutralizing antibodies against VEGF-A (R&D, 25 μg/mL) and/or SDF-1a (Abcam, 10 μg/mL) and was continuously stirred at 4 °C for 2 h to allow the resin to bind to the neutralizing antibodies.

Techniques: Tube Formation Assay, In Vitro, Incubation, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Hypoxic Preconditioning Enhances Cellular Viability and Pro-angiogenic Paracrine Activity: The Roles of VEGF-A and SDF-1a in Rat Adipose Stem Cells

doi: 10.3389/fcell.2020.580131

Figure Lengend Snippet: In vivo Matrigel plug angiogenesis assay. (A) Full view, H&E (Gross), CD31 (Gross, 200X) of the Matrigel plug one week after subcutaneous transplantation in nude mice. Gross (H&E, CD31): Scale bar = 2 mm. 200X (CD31): Scale bar = 200 μm. (B) Quantitative analysis on the number and area of CD31 positive vessels was measured. Image analysis was performed on three random fields per sample ( n = 5 samples, *denotes P < 0.05 vs. NPC-ASC CM ; # denotes P < 0.05 vs. HPC-ASC CM + anti-VEGF-A/SDF-1a).

Article Snippet: To clarify the contribution of SDF-1a and VEGF-A in HPC-ASC CM -induced angiogenesis, the HPC-ASC CM was incubated with neutralizing antibodies against VEGF-A (R&D, 25 μg/mL) and/or SDF-1a (Abcam, 10 μg/mL) and was continuously stirred at 4 °C for 2 h to allow the resin to bind to the neutralizing antibodies.

Techniques: In Vivo, Angiogenesis Assay, Transplantation Assay