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Notes: The NT-3 Emax^® ImmunoAssay System was used to examine NT-3 levels in adult rat spinal cord following intrathecal injection of siRNAs. The C₃-C₅ sections of the spinal cord were dissected after treatment and homogenized in a cold extraction buffer (Tris-buffered saline [pH 8.0], 1% NP-40, 10% glycerol, 5mM sodium metavanadate, 10mM PMSF, 100μg/ml aprotinin and 10μg/ml leupeptin).  The homogenates were acid treated, cleared by centrifugation, and then analyzed with the NT-3 Emax^® ImmunoAssay System.  Results were normalized by a protein assay. The researchers also used Promega’s recombinant human BDNF for phrenic activity studies on rats.  In these experiments BDNF was dissolved in artificial cerebral spinal fluid containing 0.1%BSA.   (3043)

Notes: The anti-TrkB In polyclonal antibody was used to immunohistochemically stain brains of 1 to 4 day old mice. The researchers used a 1:100 dilution of the antibody on 20-30μm thick brain sections. Sections were stained in three steps with biotin labeled anti-chicken antibody, a streptavidin-HRP conjugate, and an Alexa Fluor 488 tyramide dye that is preferentially reduced by HRP (Molecular Probes). Details of the entire procedure are provided.  BDNF and NGF were also used in in vitro neuron studies to examine neuron respiratory frequency.  For these experiments the researchers added 100ng/ml of each neurotrophic factor to disected pre-Bötzinger complexes (PBCs).  (2764)

Notes: The factors BDNF and NT-3 were used to maintain cultures. RNasin^® Ribonuclease Inhibitor and Taq DNA Polymerase were used for RT-PCR of rat total RNA derived from arcuate and periventricular cultures from newborn rats as well as intermediate lobe cells from adult rats. (0745)

Notes: Recombinant Human BDNF was used in studies of BDNF neutralization. The Anti-BDNF pAb (> or = 10µg/ml) was found to inhibit 50ng/ml of BDNF from activating autophosphorylation cascades of TrkB in TrkB-expressing NIH3T3 cells. Control IgY at up to 40µg/ml had no inhibitory effect on the autophosphorylation. The Anti-BDNF pAb was added to cultures of primary rat sympathetic neurons to neutralize any BDNF/p75NTR autocrine loop. BDNF causes a decrease in cell proliferation and apoptosis as judged by the CellTiter 96^® Assay and the DeadEnd™ Colorimetric Apoptosis Detection System, respectively. The DeadEnd™ System was used in a unique way. Rather than staining the apoptotic nuclei with DAB via the streptavidin-HRP conjugate, a Cy^®3-conjugated streptavidin was substituted. To compare the immunolocalization of the tyrosine hydroxylase protein and the p75NTR receptor, serial sections of mouse pineal glands were probed with an anti-tyrosine hydroxylase antibody and the Anti-Human p75 pAb. A lot of detail is provided for tissue processing prior to IHC. The Anti-Human p75 pAb was also used to detect p75 in mouse and rat pineal gland extracts via Western blotting. The name of the DeadEnd™ Colorimetric Apoptosis Detection System has been changed to DeadEnd™ Colorimetric TUNEL System. (0916)

Notes: To test the efficacy of TrkA, B and C-IgG fusions to their respective ligands, PC12 cells were stimulated Nerve Growth Factor (NGF) and TrkA-IgG, TrkB-IgG, or TrkC-IgG. Only the TrkA-IgG blocked tyrosine phosphorylation as judged by a pan phosphoTrk antibody. The TrkB-IgG blocked the activity of Brain-Derived Neurotrophic Factor (BDNF) on cortical cells and TrkC-IgG blocked Neurotrophin-3 (NT-3) action. Promega's BDNF, NT-3 and NGF, 2.5S, were used. (1452)

Notes: The BDNF E_max^® ImmunoAssay System was used to determine the levels of BDNF in the conditioned media of primary rat cerebellar neurons. The conditioned media (4ml) was concentrated (200µl) using a Centricon-10 Microconcentrator prior to assay. Cells treated with with N-methyl-D-aspartate released more BDNF than control cells at all time points. After 3 hours of treatment, the granule cells alone released 84.0 ± 9.3pg/ml and the treated cells released 151.3 ± 31. The BDNF released into the media was detectable by Western blotting with an Anti-Human BDNF pAb (antibody attributed to Promega but not mentioned in the Materials and Methods). The cultured granule cells were also assayed for TrkB phosphorylation by incubating with Recombinant Human BDNF. (0733)

Notes: The Recombinant Human Brain-Derived Neurotrophic Factor was used in studies of quantal amplitude on pyramidal neurons. (0467)

Notes: Recombinant Human Brain-derived Neurotrophic Factor (rhBDNF) was used on postnatal rat visual cortex cultures to study the BDNF-dependent regulation of cortical inhibition. (0466)

Notes: Both Recombinant Human Brain-Derived Neurotrophic Factor (BDNF) and Glial Cell-Derived Neurotrophic Factor (GDNF) were assayed for protection of midbrain cultures from the neurotoxin, MPP+. (0688)

Notes: Recombinant Human BDNF was used on hippocampal slice cultures from trkB(-/-) mice. The effects on cell survival due to the neurotrophin were examined. (1505)

Notes: rhBDNF was used to assay the ability of neurotrophins to support the survival of spiral ganglion neurons in culture alone or in combination with other factors. (1578)