Journal: Neural Regeneration Research

Article Title: Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury ★

doi: 10.3969/j.issn.1673-5374.2012.10.004

Figure Lengend Snippet: Neurotrophic factor expression in surrounding injured brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. (A–C) Nerve growth factor (NGF) protein expression (immunohistochemistry, × 200); (D–F) brain-derived neurotrophic factor (BDNF) protein expression (immunohistochemistry, × 200); (G–I) BDNF mRNA expression ( in situ hybridization, × 400). (A, D, G) Results (absorbance) are expressed as mean ± SD from six rats in each group at each time point. a P < 0.05, vs . model group ( t -test was used to specify differences between two groups at the corresponding time points); (B, E, H) model group at 14 days after transplantation; (C, F, I) transplantation group at 14 days after transplantation. Arrows: NGF protein-, BDNF protein-, and BDNF mRNA-positive cells.

Article Snippet: Primary antibodies were incubated overnight at 4°C in wet box with the following dilutions: rat anti-human BrdU monoclonal antibody (1:150; Beijing Boaosen Biotechnology, China), rabbit anti-mouse BDNF (1:150; Boster, Wuhan, China), NGF (1:150; Boster), VEGF (1:150; Boster), CD34 (1:100; Boster), bFGF (1:100; Beijing Boaosen Biotechnology) polyclonal antibody.

Techniques: Expressing, Transplantation Assay, Immunohistochemistry, Derivative Assay, In Situ Hybridization

Journal: Cell Proliferation

Article Title: Flavonoid chrysin activates both TrkB and FGFR1 receptors while upregulates their endogenous ligands such as brain derived neurotrophic factor to promote human neurogenesis

doi: 10.1111/cpr.13732

Figure Lengend Snippet: Resource of antibodies.

Article Snippet: Rabbit anti BDNF , ABclonal , A16299.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Plasmid map of the tetracycline-regulatable retroviral expression vector pLN–tet-on and timeline of in vivo experiments. A, Expression of the reversed tetracycline transactivator (rtTA2s–M2) is driven by the 5′ long terminal repeat (5′LTR), and expression of the neomycin resistance gene (neo) is driven by an internal SV40 promoter (SV40). Note that the tet-responsive minimal cytomegalovirus promoter (CMV*−1) is oriented in the opposite orientation to the 5′LTR. cDNAs for BDNF and GFP, respectively, are inserted into the multiple cloning site. Two polyadenylation signals (polyA) are present, one located in the 3′LTR and a bovine growth hormone polyA signal behind the multiple cloning site for the inducible transcript. B, Survival and treatment of animals for histological and ELISA analysis. Duration of treatment with doxycycline in the drinking water is indicated by plus symbols (Dox++++), and survival without doxycycline treatment is indicated by the dashed line (No Dox—-). Note that several groups were first treated with doxycycline followed by doxycycline removal (++++ followed by —-). The number of animals used for histological analysis are indicated at each time point for each treatment group, and numbers in parentheses indicate animal numbers used for BDNF ELISAs.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Plasmid Preparation, Retroviral, Expressing, In Vivo, Cloning, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Dose dependence and kinetics of BDNF production in primary Fischer 344 rat fibroblasts transfected with tet-on–BDNF. A, With increasing concentrations of doxycycline in the cell culture medium, BDNF expression is increased. B, Kinetics of BDNF expression. tet-on–BDNF transfected cells were cultivated for 3 d with doxycycline (red and black lines) or without doxycycline (blue and green circles). Supernatants were collected beginning at time 0. One group (red line) was changed from doxycycline-containing to doxycycline-free medium, and one group (blue line) was changed from doxycycline-free to doxycycline-containing medium. BDNF levels were measured by ELISA. Cells that were constantly cultivated in doxycycline-free medium (green line) showed very little BDNF expression. Cells that were constantly treated with 1 μg/ml doxycycline (black line) showed continued BDNF expression. Cells that were first in doxycycline-free medium and were then changed to doxycycline-containing culture medium at time 0 (blue line) turned on gene expression within 12 h. This was evidenced by the 24 h supernatant (collected between the 12 and 24 h medium change), which contained high levels of BDNF. Cells that were first cultivated in doxycycline-containing medium and changed to doxycycline-free medium at time 0 (red line) nearly completely shut down gene expression after 24 h. Error bars in A and B are not always visible because of their small size.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Transfection, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: The Journal of Neuroscience

Article Title: Transient Growth Factor Delivery Sustains Regenerated Axons after Spinal Cord Injury

doi: 10.1523/JNEUROSCI.1903-07.2007

Figure Lengend Snippet: Regulated expression of GFP and BDNF in vivo. A, B, GFP fluorescence in grafts of genetically modified primary fibroblasts in the injured spinal cord 2 weeks after grafting. A, Animals that were treated with doxycycline (+Dox) show GFP fluorescence in the graft, whereas no GFP fluorescence can be found in animals that received GFP grafts in the absence of doxycycline administration (−Dox) (B). Host/graft (g) interface is indicated by dashed lines. C, D, Immunolabeling for GFP shows expression in grafts of animals that were treated with doxycycline for 3 months (C), whereas very weak GFP expression can be found in animals that received GFP grafts in the absence of doxycycline administration (−Dox) for 3 months (D). E, Regulated expression of BDNF in primary fibroblasts grafted to the injured spinal cord. Two weeks after grafting, GFP- and BDNF-expressing grafts were dissected from animals that were either untreated (−Dox) or treated with doxycycline in the drinking water (+Dox) to turn gene expression on. BDNF levels were determined by ELISA. tet-on–BDNF transfected fibroblast grafts from animals that were treated with doxycycline (BDNF + Dox) show significantly higher BDNF levels than untreated animals (BDNF − Dox) and animals with control grafts (***p < 0.001). BDNF levels in untreated animals with tet-on–BDNF transfected fibroblasts grafts are not significantly different from treated (p = 0.76) or untreated (p = 0.85) GFP transfected control animals. Scale bar: A, B, 283 μm; C, D, 138 μm.

Article Snippet: BDNF ELISA Ninety-six-well plates were coated with a rabbit anti-BDNF antibody (gift from Amgen, Thousand Oaks, CA) in carbonate buffer using normal rabbit IgG as control.

Techniques: Expressing, In Vivo, Fluorescence, Genetically Modified, Immunolabeling, Gene Expression, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons

doi: 10.3389/fncel.2014.00107

Figure Lengend Snippet: Two anti-BDNF antibodies enable specific and dense indirect immunolabeling of endogenous BDNF in hippocampal neurons in vitro . (A) Antibody verification with recombinant BDNF. HeLa cells stably expressing lentivirally-delivered BDNF IRES GFP were labeled with different anti (α)-BDNF antibodies from different species (r, rabbit; m, mouse; s, sheep). Antibody details are given in the Material and Method section. As transduction control, GFP was expressed bicistronically under an IRES2 sequence and postlabeled with chicken anti-GFP. BDNF was accumulated in the early secretory trafficking pathway after microtubule disruption with nocodazole, causing a perinuclear Golgi-like staining pattern. (B) Immunoblot of rαBDNF (17 h). Serial dilutions of recombinant BDNF (250–31 pg per lane) served as control (left panel). Hippocampal BDNF harvested at postnatal day 5 was absent in bdnf −/− mice, while heterozygous and wildtype controls show mature BDNF at a M r of 13 kDa. HRP-conjugated anti-Cytochrome C (Cyt C) served as loading control (left panel). In adult mice, rαBDNF (17 h) revealed mature BDNF at high concentration in the hippocampus (left and right lane), while only low amounts were detected in cerebellar protein lysates (middle lane). (right panel). Immunoblots revealed high specificity of the antibody 17 h. (C) Endogenous BDNF in hippocampal neurons at day in vitro (DIV) 35. In somatic regions, BDNF was not concentrated in perinuclear Golgi cisternae, but rather in somatic vesicles. Golgi cisternae were labeled with luminal GFP, a marker for the anterograde, secretory pathway. Luminal GFP and endogenous BDNF co-labeled anterograde trafficking structures (arrow heads) in neurites of hippocampal neurons. Endogenous BDNF was double-labeled by two different anti-BDNF antibodies (rabbit anti-BDNF 17h and mouse anti-BDNF mab9). (D) Somatic anti-BDNF labels were lost when the bdnf gene was removed from single cells. Hippocampal neurons from bdnf fl/fl mice were transduced with a low titer of a lentivirus expressing tdTomato IRES Cre. Neurons (DIV 20) were labeled with anti-BDNF (mab9). Tomato+ cells (upper neuron, red, yellow arrow) lacked a typical vesicular somatic BDNF label, while untransduced, Tomato-Cre-deficient neurons (lower neuron, cyan arrow) exhibited a strong somatic BDNF label. In the composite image Tomato (red) and mouse anti-BDNF (mab9) (green) labels are shown together with an anti-neurofilament (Nfh) label (white) and a nuclear DAPI stain (blue). Bar: 25 μm.

Article Snippet: Blocking and antibody incubation were performed in 10% goat serum, 5% milk powder (Biorad) for 3–4 h. For BDNF detection, rabbit anti-BDNF 17H (1/6000, provided by Michael Sendtner, Institute for Clinical Neurobiology, Würzburg, Germany) was incubated over night at 4°C.

Techniques: Immunolabeling, In Vitro, Recombinant, Stable Transfection, Expressing, Labeling, Transduction, Control, Sequencing, Disruption, Staining, Western Blot, Concentration Assay, Marker

Journal: Neurochemistry international

Article Title: DL-3-n-butylphthalide Induced Neuroprotection, Regenerative Repair, Functional Recovery and Psychological Benefits following Traumatic Brain Injury in Mice

doi: 10.1016/j.neuint.2017.03.017

Figure Lengend Snippet: TBI mice received acute as well as daily TBI intranasal treatments until sacrifice. Expressions of regenerative factors were measured using Western blot 21 days after TBI. A. The protein levels of BDNF, VEGF, GDNF, eNOS, CXCR4, and MMP-9. B. Quantified data from A. NBP treatments significantly enhanced the expression of BDNF, VEGF, eNOS, and MMP-9 compared to the control group. There was no significant change in GDNF and CXCR4 expressions among groups. * P<0.05 versus sham group; # P<0.05 versus TBI-saline control group by one-way ANOVA followed by Bonferroni correction; n=5 per group.

Article Snippet: The primary antibodies used and the dilutions for each were rabbit anti-cleaved caspase-3 antibody (Cell Signaling, Danvers, MA) at 1:400, rabbit anti-caspase-9 antibody (Cell Signaling) at 1: 1,000, rabbit anti-cytochrome c antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:200 or rabbit anti-AIF antibody (Cell Signaling) at 1:500, rabbit anti-TNF-α (Cell Signaling) at 1:1000, rabbit anti-IL-1β (Cell Signaling) at 1:1000, rabbit anti-BDNF (BD Bioscience, San Jose, CA) at 1:2,000, rabbit anti-VEGF (BD Bioscience) at 1,000, rabbit anti-GDNF (BD Bioscience) at 1:1,000, rat anti-eNOS (BD Bioscience) at 1:1,000, rabbit anti-CXCR4 (BD Bioscience) at 1:2,000, rabbit anti-p65 (Cell Signaling) at 1:1,000, mouse anti-IκB (Cell Signaling) at 1:1,000, mouse anti-p-IκB (Cell Signaling) at 1:1,000, rabbit anti-COX (Cell Signaling) at 1:2,500, mouse anti-actin (Sigma-Aldrich) at 1:5000, and rabbit anti-MMP-9 antibody (Millipore, Billerica, MA) at 1:2500.

Techniques: Western Blot, Expressing