cntf duoset elisa kit  (RayBiotech inc)

Journal: British journal of pharmacology

Article Title: Neurotrophic interactions between neurons and astrocytes following AAV1-Rheb(S16H) transduction in the hippocampus in vivo.

doi: 10.1111/bph.14882

Figure Lengend Snippet: FIGURE 4 Elevation of CNTF production in hippocampal astrocytes via activation of the BDNF/TrkB/mTORC1 signalling pathway. (a) Immunohistochemical staining showing increased CNTF expression in glia‐like cells of the hippocampal CA1 region of CON, AAV1‐GFP‐ injected, and AAV1‐Rheb(S16H)‐injected rats (black arrows). Scale bars, 500 μm (inset 20 μm). (b) Double immunofluorescence staining against GFAP (green) and CNTF (red) showing the co‐localization of these two markers in the CA1 region of the hippocampus of CON and AAV1‐Rheb (S16H)‐injected rats. Scale bars, 100 μm (inset 10 μm). (c, d) Western blot analysis of the levels of CNTF expression in the hippocampus of AAV1‐ GFP‐injected or AAV1‐Rheb(S16H)‐injected rats, with or without BDNF neutralizing antibody (B.NA) or TrkB neutralizing antibody (T.NA). Differences among groups were evaluated by Kruskal–Wallis test (c) or one‐way ANOVA (d) and Tukey's post hoc analysis. *P < .05 versus CON and #P < .05 versus Rheb(S16H) alone (n = 5 for each group). (e) Immunohistochemical staining showing increased CNTF expression in glia‐like cells of the hippocampal CA1 region of recombinant BDNF‐injected rats (black arrows). Double immunofluorescence staining against GFAP (green) and CNTF (red) showing the co‐localization of these two markers in the CA1 region of the hippocampus of recombinant BDNF‐injected rats. Scale bar, 50 μm. (f) Western blot analysis of CNTF. Differences between groups were evaluated by Student's unpaired t test. *P < .05 versus CON (n = 5 for each group). (g–i) Measurement of CNTF concentration in the conditioned medium (CM) of recombinant BDNF‐treated astrocyte cultures using ELISA kits. (g) Schematic of the experimental design for measuring the levels of CNTF in hippocampal astrocyte cultures. (h) Histogram showing the dose‐dependent effects of recombinant BDNF on CNTF release in astrocyte cultures. Differences among groups were evaluated by Kruskal–Wallis test and Tukey's post hoc analysis. *P < .05 versus CON (n = 5 for each group). (i) Quantitative results showing the CNTF concentration after the treatment of astrocyte cultures with recombinant BDNF (80 ng·ml−1), with or without GNF‐5837 (10 μM) or rapamycin (80 nM). Differences among groups were evaluated by one‐way ANOVA and Tukey's post hoc analysis. *P < .05 versus CON and #P < .05 versus recombinant BDNF alone (n = 5 for each group)

Article Snippet: To quantify the CNTF released in the BDNF‐treated astrocyte culture medium, we used commercially available ELISA kits according to the manufacturer's protocol (Cat No. DY557; R&D Systems).

Techniques: Activation Assay, Immunohistochemical staining, Staining, Expressing, Injection, Double Immunofluorescence Staining, Western Blot, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Neural Regeneration Research

Article Title: Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

doi: 10.4103/1673-5374.180744

Figure Lengend Snippet: Expression of endogenous CNTF mRNA and protein in normal and the denervated hippocampi 7 days after right FiFx transection. (A) mRNA levels of CNTF (161 bp) and GAPDH (452 bp, as a reference) detected by RT-PCR. (B) Quantification of CNTF mRNA. Relative expression was expressed as the optical density ratio of CNTF to GAPDH. (C) Western blot assay of protein levels of CNTF and β-actin (as a reference). The molecular weight of CNTF and β-actin are 24 kDa and 42 kDa, respectively. (D) Quantification of CNTF protein. Relative expression was expressed as the optical density ratio of CNTF to β-actin. (E) ELISA identified CNTF protein levels in normal and denervated hippocampi. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, vs . normal hippocampus (two-tailed Student's t -test, n = 3). CNTF: Ciliary neurotrophic factor; RT-PCR: reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ELISA: enzyme linked immunosorbent assay; FiFx: fimbria-fornix.

Article Snippet: Supernatants were harvested and the amount of CNTF was determined using a rat CNTF ELISA kit (R&D, Minneapolis, MN, USA), according to the manufacturer's instruction.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

doi: 10.4103/1673-5374.180744

Figure Lengend Snippet: Immunofluorescence analysis of MAP2 and GFAP expression in hippocampal NSCs derived from embryonic day 17 fetal rats. (A) Cells were separately stained for GFAP (red) and MAP2 (green), with total cell number indicated by Hoechst (blue). Scale bar: 20 µm. (B) MAP2-positive cells are indicated by a percentage of total cells. (C) GFAP-positive cells are indicated by a percentage of total cells. Data are the mean ± SEM. Experiments were performed in triplicate. * P < 0.05, ** P < 0.01, vs . control (two-tailed Student's t -test). MAP2: Microtubule associated protein-2; GFAP: glial fibrillary acidic protein; CNTF: ciliary neurotrophic factor.

Article Snippet: Supernatants were harvested and the amount of CNTF was determined using a rat CNTF ELISA kit (R&D, Minneapolis, MN, USA), according to the manufacturer's instruction.

Techniques: Immunofluorescence, Expressing, Derivative Assay, Staining, Control, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Schwann Cells But Not Olfactory Ensheathing Cells Inhibit CNS Myelination via the Secretion of Connective Tissue Growth Factor

doi: 10.1523/JNEUROSCI.3233-13.2013

Figure Lengend Snippet: The expression of CNTF, and CTGF in SCs and OECs. A, qRT-PCR analysis of purified SCs and OECs demonstrated the significant upregulation of CTGF mRNA in SCs compared with OECs. B, ELISA demonstrated greater levels of CTGF in SCM compared with OCM and ACM and heat-treated SCM (h.SCM). There was no significant difference in secreted levels of CTGF comparing ACM, OCM, and h.SCM. C, ACM contained the greatest amount of the promyelinating factor CNTF, compared with OCM or SCM. There were no significant differences in CNTF levels in SCM versus OCM. *p < 0.05. **p < 0.01. n = 3 biological replicates for qRT-PCR; n = 4 batches of CM for ELISA.

Article Snippet: To assess the levels of CNTF or CTGF in CM from SCs, OECs, and astrocytes, ELISA kits were used according to the manufacturer's instructions (CNTF DuoSet ELISA Kit, RayBiotech; CTGF standard protein detection kit, PeproTech).

Techniques: Expressing, Quantitative RT-PCR, Purification, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Impaired Expression of Ciliary Neurotrophic Factor in Charcot-Marie-Tooth Type 1A Neuropathy

doi: 10.1097/nen.0b013e31819fa6ba

Figure Lengend Snippet: FIGURE 1. Enzyme-linked immunosorbent assay quantification of ciliary neurotrophic factor (CNTF) in sciatic nerves. (A) CNTF was significantly downregulated in heterozygous Charcot-Marie-Tooth disease type 1A (CMT1A) rats compared with their wild- type (Wt) littermates (mean T SEM: 230.4 T 29.30 vs 689.3 T 43.25 pg; n = 4; p G 0.0001). (B) CNTF expression was significantly lower in mice that overexpress peripheral myelin protein 22 (Pmp22) (Pmp22tg), another animal model for CMT1A (mean T SEM: 590 T 33.50 vs 3,605 T 45 pg; n = 4; p G 0.0001). CNTF levels were unchanged or even increased in sciatic nerves of Pmp22j/j

Article Snippet: CNTF content was determined by enzyme linked immunosorbent assay using a Rat CNTF DuoSet kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Animal Model

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Impaired Expression of Ciliary Neurotrophic Factor in Charcot-Marie-Tooth Type 1A Neuropathy

doi: 10.1097/nen.0b013e31819fa6ba

Figure Lengend Snippet: FIGURE 2. Ciliary neurotrophic factor (CNTF) expression in vivo: sciatic nerve crush experiments and immunohistochemistry. (A, B) Cntf real-time reverse transcriptase (RT) polymerase chain reaction on distal stumps of wild-type and Charcot-Marie-Tooth disease type 1A (CMT1A) rats. In unlesioned nerves, relative levels of Cntf mRNA were higher in wild-type animals (Wt [UL]) than in the transgenic ones (CMT1A [UL]) both 1 week (1 w) (mean T SEM: 15.27 T 2.567 vs 3.704 T 0.9295; n = 3; p G 0.05) and 3 months (3 m) (mean T SEM: 5.755 T 1.018 vs 2.290 T 0.4711; n = 3; p G 0.05) after the crush lesion. One week after the crush lesion, Cntf levels dramatically decreased both in wild-type (mean T SEM: 0.0398 T 0.0325) and CMT1A rats (mean T SEM: 0.0531 T 0.0147). After a recovery period of 3 months, both control and CMT1A rats reexpressed Cntf mRNA, but the levels in CMT1A animals were extremely low (mean T SEM: 2.659 T 0.7975 and 1.341 T 0.2370, respectively). (C, D) Relative levels of CNTF protein were significantly higher in wild-type compared with CMT1A rats in unlesioned nerves both at the 1 w (mean T SEM: 307.6 T 1.732 vs 53.4 T 1.674 pg; n = 3; p G 0.0001) (C) and the 3 m (mean T SEM: 198 T 26.50 vs 57.67 T 8.603 pg; n = 3; p G 0.05) (D) time points. (C) One week after the crush lesion, CNTF levels decreased both in wild-type (mean T SEM: 174.4 T 28.23 pg) and transgenic rats (mean T SEM: 15.9 T 6.062 pg). (D) At 3 m, increased levels of CNTF compared with the baseline were found in wild-type and in CMT1A rats (mean T SEM: 430 T 129.8 pg and 94 T 16.11 pg, respectively), but the levels of CNTF in CMT1A nerves were still significantly lower than in the controls. Data were analyzed by a two-tailed t-test for unpaired samples. (E, F) Immunohistochemistry of CNTF on unlesioned rat sciatic nerves confirmed underexpression of this cytokine in myelinating SCs of CMT1A rat nerves (E) compared with their wild-type littermates (F). Arrows indicate Schwann cell staining. 2,3-Diaminobenzidine; scale bars = 100 Km.

Article Snippet: CNTF content was determined by enzyme linked immunosorbent assay using a Rat CNTF DuoSet kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Expressing, In Vivo, Immunohistochemistry, Reverse Transcription, Polymerase Chain Reaction, Transgenic Assay, Control, Two Tailed Test, Staining

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Impaired Expression of Ciliary Neurotrophic Factor in Charcot-Marie-Tooth Type 1A Neuropathy

doi: 10.1097/nen.0b013e31819fa6ba

Figure Lengend Snippet: FIGURE 4. Ciliary neurotrophic factor (CNTF) and neuronal proteins. (A, B) Enzyme-linked immunosorbent assay quantification of CNTF in Charcot-Marie-Tooth disease type 1A (CMT1A) rats at different time points. (A) At all time points, transgenic rats () express significantly lower levels of CNTF compared with wild-type littermates (O) (mean T SEM at P3: 82.9 T 0.65 vs 108.9 T 3.15 pg; n = 4; p G 0.001. P30: 229.9 T 30.7 vs 675.6 T 115.2 pg; n = 4; p G 0.01. P320: 152.6 T 11.1 vs 684.4 T 140.7 pg; n = 4; p G 0.01). Expression of CNTF protein in CMT1A nerve as a percentage of wild-type levels (B) demonstrates that the levels of CNTF in affected animals decrease with increasing age. (C, D) Western blot analysis of nonphosphorylated neurofilaments (SMI32) and amyloid precursor protein (APP). The levels of nonphosphorylated neurofilaments (NFs) (C) were similar in CMT1A (+/j; white bars) and wild-type (Wt; black bars) rats (mean T SEM: 3,216 T 405.5 vs 3,275 T 97.17; n = 3; p = ns) at P3. Paralleling the reduction in CNTF expression, at both P30 and P320, there were significant increases of nonphosphorylated NFs in transgenic compared with normal control rats (mean T SEM at P30: 3,932 T 256.2 vs 3,037 T 95.49; n = 3; p G 0.05) (mean T SEM at P320: 4,708 T 423.3 vs 2,918 T 261.5; n = 3; p G 0.05). Amyloid precursor protein A4 (D) was also not significantly different between CMT1A (+/j; white bars) and wild-type (Wt; black bars) rats at P3 (mean T SEM: 1,802 T 541.6 vs 2,501 T 259.8; n = 3; p = ns) and P30 (mean T SEM: 1,637 T 94.69 vs 2,086 T 288.1; n = 3; p = ns), but at P320, when differences in CNTF and nonphosphorylated NFs were highest between CMT1A and normal controls, APP was also significantly increased in CMT1A rats (mean T SEM: 5,861 T 302.5 vs 2,906 T 541.0; n = 3; p G 0.01). Data were analyzed by a two-tailed t-test for unpaired samples.

Article Snippet: CNTF content was determined by enzyme linked immunosorbent assay using a Rat CNTF DuoSet kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Transgenic Assay, Expressing, Western Blot, Control, Two Tailed Test

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Impaired Expression of Ciliary Neurotrophic Factor in Charcot-Marie-Tooth Type 1A Neuropathy

doi: 10.1097/nen.0b013e31819fa6ba

Figure Lengend Snippet: FIGURE 5. Signaling cascade downstream of ciliary neurotrophic factor (CNTF) receptor. (A, B) Representative immunoblots (IBs). Western blot analyses of STAT3 expression show that in both homozygous (+/+) and heterozygous (+/j) Charcot-Marie- Tooth disease type 1A (CMT1A) sciatic nerves (A) and Schwann cells (SCs) (B), STAT3 is overexpressed compared with the wild- type (wt) controls (sciatic nerves: mean T SEM: 21.51 T 3.984 and 7.76 T 1.963, respectively, vs 4.5 T 2.367; n = 3; analysis of variance [ANOVA], p G 0.01 and p = ns, respectively) (SCs: mean T SEM: 42.50 T 17.50 and 38.50 T 3.50, respectively, vs 17.50 T 2.50; n = 3; ANOVA, p = ns and p G 0.05, respectively) despite low levels of CNTF, but in accordance with Cntf receptor complex upregulation. Moreover, there is significantly increased activation of STAT3 (pSTAT3) in +/+ and +/j CMT1A rats (A) and SCs (B) compared with wt controls (sciatic nerves: mean T SEM: 23.73 T 2.309 and 7.96 T 1.097, respectively, vs 1.1 T 0.3464; n = 3; ANOVA, p G 0.001 and p G 0.01, respectively) (SCs: mean T SEM: 17.50 T 2.50 and 22.50 T 2.50, respectively, vs 0.75 T 0.25; n = 3; ANOVA, p G 0.05 for both comparisons). As a result, there is increased STAT3 tyrosine phosphorylation (pSTAT3/ STAT3) in both +/+ and +/j CMT1A rats (A1) and SCs (B1) compared with the wt controls (pSTAT3/STAT3 in sciatic nerves: mean T SEM: 1.3 T 0.1732 and 1.1 T 0.6 vs 0.3464 T 0.1155; n = 3; ANOVA, p G 0.05 and p = ns, respectively) (pSTAT3/STAT3 in SCs: mean T SEM: 0.4650 T 0.1350 and 0.5800 T 0.01 vs 0.12 T 0.02; n = 3; ANOVA, p = ns and p G 0.05, respectively).

Article Snippet: CNTF content was determined by enzyme linked immunosorbent assay using a Rat CNTF DuoSet kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Activation Assay, Phospho-proteomics

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Impaired Expression of Ciliary Neurotrophic Factor in Charcot-Marie-Tooth Type 1A Neuropathy

doi: 10.1097/nen.0b013e31819fa6ba

Figure Lengend Snippet: FIGURE 8. Myelinating dorsal root ganglia (DRG) cultures were treated with rat recombinant ciliary neurotrophic factor (rrCNTF) (0.5 ng/mL) for 21 days. Panel A: Dose-response experiments were performed on Charcot-Marie-Tooth disease type 1A (CMT1A) DRG cultures to test the effects of different amounts of rrCNTF on the expression of nonphosphorylated neurofilaments (SMI32) as a marker of axonal injury. In the range tested (0.1Y10 ng/mL), the best effect was obtained for 0.5 ng/mL. Panel B: (A) Administration of rrCNTF to CMT1A cultures did not improve the rate of myelination in any of the treated cultures (mean number of myelinated segments/mm2 T SEM: 0.000193 T 0.00003; n = 10 in vehicle treated cultures vs 0.000207 T 0.00003; n = 10 in rrCNTF-treated cultures; p = ns). Representative cultures are illustrated on the left. (B) Graph of Western blot analysis of SMI32 shows that chronic treatment with rrCNTF reduced this axon injury marker compared with vehicle-treated cultures. On average, expression of SMI32 was significantly reduced by treatment with rrCNTF in transgenic cultures from 3 different offspring (vehicle- treated: 1.154 T 18.35 vs treated: 0.93 T 0.65; n = 20; p G 0.05). Treatment of wild-type cultures with rrCNTF did not affect expression of SMI32 (0.59 T 0.3 vs 0.56 T 0.4; n = 7; p = ns). (Osmium tetroxide and Sudan black; scale bars = 10 Km). Data were analyzed by a two-tailed t-test for paired samples.

Article Snippet: CNTF content was determined by enzyme linked immunosorbent assay using a Rat CNTF DuoSet kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Marker, Western Blot, Transgenic Assay, Two Tailed Test