rabbit polyclonal anti nav 1 7  (Alomone Labs)

Journal: Journal of Neuroinflammation

Article Title: Functional up-regulation of Na v 1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

doi: 10.1186/1742-2094-11-45

Figure Lengend Snippet: Cellular distribution of Na v 1.8 in rat DRG neurons. (A–F) Immunohistochemical distribution of Na v 1.8 in DRG neurons isolated from sham and CFA-treated rats. Panels show the localization of Na v 1.8 (green) in NF200-positive neurons (red, arrowheads). Yellow signal indicates double-labeled neurons. After inflammation, numerous large NF200-positive ganglion cells co-express Na v 1.8 (arrows). (G) Proportion of Na v 1.8-immunoreactive neurons in lumbar DRGs. The number of neurons expressing Na v 1.8 increases in CFA-inflamed rats compared to sham. (H) Percentage of NF200-containing DRG neurons expressing Na v 1.8. The proportion of dually stained cells increases after CFA-induced chronic inflammation. Data in panels G and H shown as mean ± SEM. Asterisks denote a statistically significant increase as compared with sham (4 rats/group, 10 sections/rat; *** P <0.001 or ** P <0.01; ANOVA followed by Sidak’s multiple comparisons test MCT)). # Statistically different from day 3; ### P <0.001, ## P <0.01, # P <0.05). Scale bars: 200 μm in A, C, and E and 20 μm in B, D, and F .

Article Snippet: To detect Na v 1.8, sections were incubated with the rabbit polyclonal anti-Na v 1.8 antibody (1:200; Alomone Labs, Jerusalem, Israel) in PBS containing 0.05% Triton X-100 and 0.5% NGS.

Techniques: Immunohistochemical staining, Isolation, Labeling, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: Functional up-regulation of Na v 1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

doi: 10.1186/1742-2094-11-45

Figure Lengend Snippet: Changes in the expression and distribution of Na v 1.8 associated with CFA-induced inflammation in rats. (A) Na v 1.8 mRNA levels of the ipsilateral hind paw are determined by qRT-PCR for sham animals and 14 days following CFA injection. Data are expressed as mean ± SEM (6–8 rats/group). ** P <0.01, CFA alone vs. sham (unpaired Student’s t -test). (B, C) Immunohistochemical staining of Na v 1.8 channels in the rat sciatic nerve proximal to the lesion site 48 h after ligation. The ligature was placed around the sciatic nerve proximal to the trifurcation on day 12 post-CFA. Scale bar: 100 μm. (D) Accumulation of Na v 1.8-like immunoreactivity is significantly increased in 14 day post-CFA rats compared to sham animals (* P <0.05; unpaired Student’s t- test).

Article Snippet: To detect Na v 1.8, sections were incubated with the rabbit polyclonal anti-Na v 1.8 antibody (1:200; Alomone Labs, Jerusalem, Israel) in PBS containing 0.05% Triton X-100 and 0.5% NGS.

Techniques: Expressing, Quantitative RT-PCR, Injection, Immunohistochemical staining, Staining, Ligation

Journal: Journal of Neuroinflammation

Article Title: Functional up-regulation of Na v 1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

doi: 10.1186/1742-2094-11-45

Figure Lengend Snippet: Na v 1.8 currents are enhanced in large-sized DRG neurons from inflamed rats. (A) Immunofluorescence staining of Na v 1.8 (green) and NF200 (red) on acutely dissociated primary afferent neurons, 14 days post-CFA. Merge images show dually labeled large-sized sensory neurons (yellow). (B) Isolation of TTX-resistant Na v 1.8 currents in large-sized sensory neurons from sham and inflamed rats. Na v 1.8 currents are significantly increased post-CFA. Representative I-V curves of currents are determined using the pulse protocol indicated in the inset. (C) I-V curves of Na v 1.8 currents obtained from large-soma DRG neurons. The peak maximum current is observed at 0 mV in all groups, with the exception of day 14 (–10 mV). (D) Peak Na v 1.8 current densities are significantly increased at days 3, 8, and 14 post-CFA injection (*** P <0.001 * P <0.05 vs. sham; $$$ P <0.001 vs. day 3; ### P <0.001 vs. day 8; ANOVA followed by Sidak’s MCT; n = 6–13). (E, F) Kinetic properties of Na v 1.8 currents in large-sized sensory neurons. CFA treatment induces a leftward shift of the activation (E) and inactivation (F) curves of Na v 1.8 current. Half-activation and half-inactivation potentials and slope factors are summarized in Additional file : Table S3.

Article Snippet: To detect Na v 1.8, sections were incubated with the rabbit polyclonal anti-Na v 1.8 antibody (1:200; Alomone Labs, Jerusalem, Israel) in PBS containing 0.05% Triton X-100 and 0.5% NGS.

Techniques: Immunofluorescence, Staining, Labeling, Isolation, Injection, Activation Assay

Journal: Journal of Neuroinflammation

Article Title: Functional up-regulation of Na v 1.8 sodium channel in Aβ afferent fibers subjected to chronic peripheral inflammation

doi: 10.1186/1742-2094-11-45

Figure Lengend Snippet: Ambroxol treatment blocks the changes in the biophysical properties of Na v 1.8 in large-sized DRG neurons extracted from rats 14 days post-CFA. (A) I-V curves of Na v 1.8 currents obtained from sham and inflamed large sensory neurons. (B) Histogram showing the effects of different concentrations of ambroxol (20 and 100 μM) on the increased Na v 1.8 peak current induced by CFA (*** P <0.001, inflamed vs. sham large sensory neurons; ### P <0.001 compared to inflamed DRG neurons; ANOVA followed by Sidak’s MCT; n = 6–13). (C) Ambroxol also significantly inhibits the leftward shift of the activation curves of Na v 1.8 current. Half-activation potential and slope factor are summarized in Additional file : Table S3. Note that the data corresponding to sham and day 14 after CFA treatment are the same as the ones in Figure .

Article Snippet: To detect Na v 1.8, sections were incubated with the rabbit polyclonal anti-Na v 1.8 antibody (1:200; Alomone Labs, Jerusalem, Israel) in PBS containing 0.05% Triton X-100 and 0.5% NGS.

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of Aβ 1–42 exposure on Na V 1.6 protein expression and activity in primary hippocampal neurons at 10–12 DIV. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control. ( D ) Representative traces of Na + currents recorded under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. (E) Representative traces of Na + currents recorded after 5 μM Aβ 1–42 (24 h) alone, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. ( F ) Normalization of Na + current densities, at −20 mV, represented in panel D and E. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus control, # p < 0.001 versus control Aβ 1–42 . (G) Representative western blot of Na V 1.6 protein expression (top) in the presence of siNa V 1.6 (50 nM; 48 h) in primary hippocampal neurons at 12 DIV. Quantification of siNav1.6 inhibition in primary hippocampal neurons (bottom). Values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control neurons.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Expression and activity of Na V 1.6 channels in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT and Tg2576 primary hippocampal neurons after 8 and 12 DIV. ( B ) Normalization of Na + current densities at −20 mV represented in panel A. Values are expressed as mean ± SEM of current densities of 3 independent experimental sessions. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( D ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( E ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. (F) Representative confocal images displaying Na V 1.6 distribution in WT (left) and Tg2576 (right) primary hippocampal neurons after 12 DIV. Scale bars: 20 μm.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of siNa V 1.6 and anisomycin on Na V 1.6 protein expression and activity in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( B ) Representative traces of Na + currents recorded in Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( C ) Normalization of Na + current densities at −20 mV represented in panel A and B. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control WT, *** p < 0.001 versus control WT, # p < 0.001 versus control Tg2576. ( D ) Representative current tracings recorded in the gap-free mode in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( E ) Quantification of spike frequency recorded in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. # p < 0.001 versus control Tg2576. ( F ) Quantification of membrane depolarization recorded in WT and Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. # p < 0.001 versus control Tg2576

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Immunocytochemical analysis of Na V 1.6 protein expression after anisomycin treatment in Tg2576 primary hippocampal neurons at 12 DIV. ( A ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in WT (a-c) and Tg2576 primary hippocampal neurons in the absence (d-f) or in the presence (g-i) of anisomycin. Scale bars in a-i: 20 μm. ( B ) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 primary hippocampal neurons in the absence or in the presence of anisomycin. Scale bars: 5 μm. Data are expressed as mean ± SEM of values obtained from 20 cells per group in 3 independent experimental sessions. ** p < 0.01 versus WT; # p < 0.001 versus Tg2576.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Evaluation of Na V 1.6 protein expression in the hippocampus of 3-month-old WT and Tg2576 mice. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. ( D ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in the hippocampus of 3-month-old WT (a-c) and Tg2576 mice (d-f). Scale bars in a-f: 20 μm. (E) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 neurons in the hippocampus of 3-month-old mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. **p < 0.01 versus WT.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment

doi: 10.1101/711614

Figure Lengend Snippet: a MAP2 and panNa v immunofluorescence in rat hippocampal neurons incubated overnight at 9-11 DIV in DMSO, LatB, or TR100. b Smoothed panNa v fluorescence intensity line profiles (gray lines) along each neurite of the corresponding neuron in ( a ) normalized to the median value (black line). c AIS localization indices for each group (Mann-Whitney U Test). Black circles represent mean value. Box borders represent the 25 th and 75 th percentiles, whiskers represent minimum and maximum values less than 1.5x the interquartile range lower or higher than the 25 th or 75 th percentiles, respectively (Tukey style). DMSO 0.2%: n = 17, 3 independent experiments; LatB 5 µM: n = 17, 3 independent experiments; TR100 5 µM: n = 17, 3 independent experiments. * denotes statistical significance. ***: p < 0.001. Scale bar: 5 µm.

Article Snippet: Rabbit polyclonal anti-panNa v was purchased from Alomone Labs (1:200, ASC-003).

Techniques: Immunofluorescence, Incubation, Fluorescence, MANN-WHITNEY