goat anti nav1 8  (Alomone Labs)


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    Alomone Labs goat anti nav1 8
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Goat Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti nav1 8/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone"

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    Journal: Molecular Pain

    doi: 10.1177/17448069211069255

    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Figure Legend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Techniques Used: Western Blot, Immunohistochemistry

    Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).
    Figure Legend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Techniques Used:

    goat anti nav1 8  (Alomone Labs)


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  • 94

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    Alomone Labs goat anti nav1 8
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Goat Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti nav1 8/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone"

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    Journal: Molecular Pain

    doi: 10.1177/17448069211069255

    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Figure Legend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Techniques Used: Western Blot, Immunohistochemistry

    Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).
    Figure Legend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

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    anti nav1 8  (Alomone Labs)


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    Alomone Labs anti nav1 8
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nav1 8/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone"

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    Journal: Molecular Pain

    doi: 10.1177/17448069211069255

    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Figure Legend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Techniques Used: Western Blot, Immunohistochemistry

    Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).
    Figure Legend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Techniques Used:

    scn10 a  (Alomone Labs)


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    Alomone Labs scn10 a
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Scn10 A, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/scn10 a/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    scn10 a - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone"

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    Journal: Molecular Pain

    doi: 10.1177/17448069211069255

    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Figure Legend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Techniques Used: Western Blot, Immunohistochemistry

    anti nav1 8  (Alomone Labs)


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  • 94

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    Alomone Labs anti nav1 8
    ALA downregulated <t>NaV1.7</t> and <t>NaV1.8</t> expression. Notes: (A) Western blots for NaV1.7 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to GAPHD for NaV1.7. ALA treatment greatly reduced expressions of NaV1.7 (n=4 for each group, ** p <0.01, compared with NS, two-sample t -test). (B) Western blots for NaV1.8 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to β-actin for NaV1.8. ALA treatment greatly reduced expressions of NaV1.8 (n=5 for NS group, n=4 for ALA group, * p <0.05, compared with NS, two-sample t -test). Abbreviations: ALA, α-lipoic acid; DRG, dorsal root ganglion; NS, normal saline.
    Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nav1 8/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "α-lipoic acid suppresses neuronal excitability and attenuates colonic hypersensitivity to colorectal distention in diabetic rats"

    Article Title: α-lipoic acid suppresses neuronal excitability and attenuates colonic hypersensitivity to colorectal distention in diabetic rats

    Journal: Journal of Pain Research

    doi: 10.2147/JPR.S135017

    ALA downregulated NaV1.7 and NaV1.8 expression. Notes: (A) Western blots for NaV1.7 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to GAPHD for NaV1.7. ALA treatment greatly reduced expressions of NaV1.7 (n=4 for each group, ** p <0.01, compared with NS, two-sample t -test). (B) Western blots for NaV1.8 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to β-actin for NaV1.8. ALA treatment greatly reduced expressions of NaV1.8 (n=5 for NS group, n=4 for ALA group, * p <0.05, compared with NS, two-sample t -test). Abbreviations: ALA, α-lipoic acid; DRG, dorsal root ganglion; NS, normal saline.
    Figure Legend Snippet: ALA downregulated NaV1.7 and NaV1.8 expression. Notes: (A) Western blots for NaV1.7 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to GAPHD for NaV1.7. ALA treatment greatly reduced expressions of NaV1.7 (n=4 for each group, ** p <0.01, compared with NS, two-sample t -test). (B) Western blots for NaV1.8 of T13-L2 DRGs from NS- and ALA-treatment rats. Bar graph showed mean density relative to β-actin for NaV1.8. ALA treatment greatly reduced expressions of NaV1.8 (n=5 for NS group, n=4 for ALA group, * p <0.05, compared with NS, two-sample t -test). Abbreviations: ALA, α-lipoic acid; DRG, dorsal root ganglion; NS, normal saline.

    Techniques Used: Expressing, Western Blot

    rabbit anti rat nav1 8  (Alomone Labs)


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    Alomone Labs rabbit anti rat nav1 8
    (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of <t>Nav1.8-</t> and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.
    Rabbit Anti Rat Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rat nav1 8/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    rabbit anti rat nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Antisense-Mediated Knockdown of Na V 1.8, but Not Na V 1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat"

    Article Title: Antisense-Mediated Knockdown of Na V 1.8, but Not Na V 1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019865

    (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of Nav1.8- and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.
    Figure Legend Snippet: (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of Nav1.8- and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Labeling, Western Blot

    nav1 8 protein  (Alomone Labs)


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    Alomone Labs nav1 8 protein
    Protein expressions of <t>Nav1.3,</t> <t>Nav1.8,</t> and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.
    Nav1 8 Protein, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nav1 8 protein/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    nav1 8 protein - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Pretreatment with intrathecal amitriptyline potentiates anti-hyperalgesic effects of post-injury intra-peritoneal amitriptyline following spinal nerve ligation"

    Article Title: Pretreatment with intrathecal amitriptyline potentiates anti-hyperalgesic effects of post-injury intra-peritoneal amitriptyline following spinal nerve ligation

    Journal: BMC Neurology

    doi: 10.1186/1471-2377-12-44

    Protein expressions of Nav1.3, Nav1.8, and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.
    Figure Legend Snippet: Protein expressions of Nav1.3, Nav1.8, and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.

    Techniques Used: Western Blot, Inhibition, Ligation

    nav1 8  (Alomone Labs)


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    Alomone Labs nav1 8
    <t>Nav1.7</t> and <t>Nav1.8</t> expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.
    Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nav1 8/product/Alomone Labs
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    nav1 8 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia"

    Article Title: Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2013/312184

    Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.
    Figure Legend Snippet: Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Techniques Used: Injection

    Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the CFA-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) CFA-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the CFA-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.
    Figure Legend Snippet: Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the CFA-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) CFA-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the CFA-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Techniques Used: Injection

    Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan- and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan- and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan- and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.
    Figure Legend Snippet: Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan- and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan- and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan- and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.

    Techniques Used: Injection

    Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3–L5 DRGs in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups. The percentage of Nav protein levels from lumbar DRGs was presented in each group. * P < 0.05, as compared to control group. # P < 0.05; comparison between inflammation and EA groups.
    Figure Legend Snippet: Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3–L5 DRGs in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups. The percentage of Nav protein levels from lumbar DRGs was presented in each group. * P < 0.05, as compared to control group. # P < 0.05; comparison between inflammation and EA groups.

    Techniques Used:

    na v 1 8  (Alomone Labs)


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    Alomone Labs na v 1 8
    ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in  or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.
    Na V 1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Schwann cells promote sensory neuron excitability during development"

    Article Title: Schwann cells promote sensory neuron excitability during development

    Journal: bioRxiv

    doi: 10.1101/2022.10.31.514415

    ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in  or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.
    Figure Legend Snippet: ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.

    Techniques Used: Staining, Expressing

    ( Caption continued on next page .) ( A ) DESI-MS analysis of SCCM. The expected m/z of PGE 2 is 351.21770 and a m/z of 351.21824 ± 1.6 ppm was detected in the SCCM sample. ( B) SCCM and PGE 2 treatments enhanced Na V 1.6, Na v 1.7 and Na v 1.8 protein levels in DRG neurons. n = 80 to 328 cells from 1-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. Untreated condition is replotted from  , as these experiments were done concurrently. ( C ) SCCM and PGE 2 treatments did not cause a major change in protein expression of indicated genes, except a significant change in Na v 1.5 protein levels. Fluorescence levels were quantified using cellpose. Gray circles, fluorescent intensity of an individual DRG neuron for the indicated genes. n = 70 to 331 cells from 2-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. p values compare cells in a one-way ANOVA and Tukey test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
    Figure Legend Snippet: ( Caption continued on next page .) ( A ) DESI-MS analysis of SCCM. The expected m/z of PGE 2 is 351.21770 and a m/z of 351.21824 ± 1.6 ppm was detected in the SCCM sample. ( B) SCCM and PGE 2 treatments enhanced Na V 1.6, Na v 1.7 and Na v 1.8 protein levels in DRG neurons. n = 80 to 328 cells from 1-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. Untreated condition is replotted from , as these experiments were done concurrently. ( C ) SCCM and PGE 2 treatments did not cause a major change in protein expression of indicated genes, except a significant change in Na v 1.5 protein levels. Fluorescence levels were quantified using cellpose. Gray circles, fluorescent intensity of an individual DRG neuron for the indicated genes. n = 70 to 331 cells from 2-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. p values compare cells in a one-way ANOVA and Tukey test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Techniques Used: Expressing, Fluorescence

    ( A ) Cartoon depicts size fractionation and mass spectrometry steps that identified PGE 2 as the excitability-inducing molecule in SCCM. ( B ) PGE 2 treatment (1 μM, 16-28 hr) increased Na V expression in DRG neurons, similar to SCCM. n = 6 distinct biological replicates per treatment group. Bottom images: Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1-EP4) antagonists blocked the SCCM-induced transcriptional increase in Na V s (see also representative images for DMSO-treated DRG neurons in  ). n = 4-6 distinct biological replicates per treatment group. ( C) PGE 2 treatment enhanced Na V protein levels in DRG neurons. Images show immunohistochemistry for indicated genes in single representative DRG neurons that were either untreated (top) or treated with PGE 2 overnight (bottom) (see also quantification in  ). ( D and E ) Quantification of the RNAscope results shown in (B), using FishQuant. Controls were either untreated cells or cells treated with DMSO vehicle (solvent for PGE 2 ). Mean ± SEM is shown for biological replicates. p values compare biological replicates in a paired t-test (D) or mixed-effect analysis (E). ( F and G ) DRG neurons treated with PGE 2 (1 μM, 16-28 hr) fired significantly more action potentials at suprathreshold current injections and exhibited a decrease in the firing threshold, similar to SCCM. Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1–EP4) antagonists blocked the excitability-inducing effect of SCCM. DRG neurons treated with 1 μM PGD 2 , a constitutional isomer of PGE 2 , remained hypoexcitable (see also  ). Mean ± SEM is shown for cells, p values compare cells in a one-way ANOVA and Tukey test. n = 20-29 total cells from 3–7 biological replicates. Untreated and SCCM conditions are replotted from  and  , as these experiments were done concurrently. ( H-I ) Injection of dmPGE 2 into the P0 sciatic nerve increased Na V 1.7 and Na V 1.8 transcript levels in lumbar DRG neurons compared to controls (the third fluorescence channel was reserved for Neun ; see  ). Results were quantified using FishQuant (I). Gray circles, Na V mRNA count in a single DRG neuron; colored circles, the average of cells in each biological replicate. Mean ± SEM is shown for biological replicates. p values compare biological replicates in cells in a one-way ANOVA and Tukey test. n = 3 mice per treatment group (at least 45 DRG neurons per mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
    Figure Legend Snippet: ( A ) Cartoon depicts size fractionation and mass spectrometry steps that identified PGE 2 as the excitability-inducing molecule in SCCM. ( B ) PGE 2 treatment (1 μM, 16-28 hr) increased Na V expression in DRG neurons, similar to SCCM. n = 6 distinct biological replicates per treatment group. Bottom images: Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1-EP4) antagonists blocked the SCCM-induced transcriptional increase in Na V s (see also representative images for DMSO-treated DRG neurons in ). n = 4-6 distinct biological replicates per treatment group. ( C) PGE 2 treatment enhanced Na V protein levels in DRG neurons. Images show immunohistochemistry for indicated genes in single representative DRG neurons that were either untreated (top) or treated with PGE 2 overnight (bottom) (see also quantification in ). ( D and E ) Quantification of the RNAscope results shown in (B), using FishQuant. Controls were either untreated cells or cells treated with DMSO vehicle (solvent for PGE 2 ). Mean ± SEM is shown for biological replicates. p values compare biological replicates in a paired t-test (D) or mixed-effect analysis (E). ( F and G ) DRG neurons treated with PGE 2 (1 μM, 16-28 hr) fired significantly more action potentials at suprathreshold current injections and exhibited a decrease in the firing threshold, similar to SCCM. Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1–EP4) antagonists blocked the excitability-inducing effect of SCCM. DRG neurons treated with 1 μM PGD 2 , a constitutional isomer of PGE 2 , remained hypoexcitable (see also ). Mean ± SEM is shown for cells, p values compare cells in a one-way ANOVA and Tukey test. n = 20-29 total cells from 3–7 biological replicates. Untreated and SCCM conditions are replotted from and , as these experiments were done concurrently. ( H-I ) Injection of dmPGE 2 into the P0 sciatic nerve increased Na V 1.7 and Na V 1.8 transcript levels in lumbar DRG neurons compared to controls (the third fluorescence channel was reserved for Neun ; see ). Results were quantified using FishQuant (I). Gray circles, Na V mRNA count in a single DRG neuron; colored circles, the average of cells in each biological replicate. Mean ± SEM is shown for biological replicates. p values compare biological replicates in cells in a one-way ANOVA and Tukey test. n = 3 mice per treatment group (at least 45 DRG neurons per mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Techniques Used: Fractionation, Mass Spectrometry, Expressing, Incubation, Immunohistochemistry, Injection, Fluorescence

    ( Caption continued on next page .) ( A to D ) RNAscope shows expression of Na V 1.6 , Na V 1.7 and Na V 1.8 transcripts in DRG neurons treated with SCCM from Flox ( Ptges3 fl/fl ) or cKO ( Ptges3 fl/fl ;Dhh CRE/+ ) mice. Micrographs of SCCM treatment from cHET ( Ptges3 fl/+ ;Dhh CRE/+ ), Dhh CRE/+ and WT mice are not shown but are also quantified in ( E to G ). ( E to G ) Conditioned media from Ptges3 conditional knockout Schwann cells (cKO) failed to enhance neuronal Na V 1.8 expression, contrary to Schwann cells collected from controls (Flox, Dhh CRE/+ , or WT). We detected a slight but significant change in Na V 1.6 and Na V 1.7 expression in DRG neurons following the addition of cKO media; however, this effect was lower than the fold increase observed with WT, Flox, or Dhh CRE/+ SCCM addition. The addition of PGE 2 to cKO media rescued the loss of Na v expression-inducing effect in cKO SCCM. Gray circles, mRNA number of an individual DRG neuron for the indicated genes. Mean ± SEM of all cells; p values compare cells in a one-way ANOVA and Tukey test. Significant increases in comparison to untreated DRG neurons or between groups indicated by brackets are shown. n = 61–195 DRG neurons from 2–4 biological replicates per group. p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
    Figure Legend Snippet: ( Caption continued on next page .) ( A to D ) RNAscope shows expression of Na V 1.6 , Na V 1.7 and Na V 1.8 transcripts in DRG neurons treated with SCCM from Flox ( Ptges3 fl/fl ) or cKO ( Ptges3 fl/fl ;Dhh CRE/+ ) mice. Micrographs of SCCM treatment from cHET ( Ptges3 fl/+ ;Dhh CRE/+ ), Dhh CRE/+ and WT mice are not shown but are also quantified in ( E to G ). ( E to G ) Conditioned media from Ptges3 conditional knockout Schwann cells (cKO) failed to enhance neuronal Na V 1.8 expression, contrary to Schwann cells collected from controls (Flox, Dhh CRE/+ , or WT). We detected a slight but significant change in Na V 1.6 and Na V 1.7 expression in DRG neurons following the addition of cKO media; however, this effect was lower than the fold increase observed with WT, Flox, or Dhh CRE/+ SCCM addition. The addition of PGE 2 to cKO media rescued the loss of Na v expression-inducing effect in cKO SCCM. Gray circles, mRNA number of an individual DRG neuron for the indicated genes. Mean ± SEM of all cells; p values compare cells in a one-way ANOVA and Tukey test. Significant increases in comparison to untreated DRG neurons or between groups indicated by brackets are shown. n = 61–195 DRG neurons from 2–4 biological replicates per group. p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Techniques Used: Expressing, Knock-Out

    ( A to H ) RNAscope and immunostaining of NaV expression in vivo in Ptges3-Flox or Ptges3-cKO mice. Left panels: RNAscope images show expression of Na V 1.7 and Na V 1.8 transcripts in lumbar DRG neurons at P0 (A) and P28 (E) (see also  ,  , and  for E16 time point and supporting data). Number of Na V 1.7 and Na V 1.8 mRNAs per cell quantified using FishQuant from P0 (B) and P28 (D). n = 4-9 mice per group. Right panels: immunohistochemistry for Na V 1.7 and Na V 1.8 in lumbar DRG neurons at P0 (C) and P28 (G). Cellular fluorescence was quantified using Cellpose. Gray circles, mRNA count or fluorescence intensity in a DRG neuron; pink circles, the average of cells in each mouse. n = 2-5 mice per group. Mean ± SEM is shown for biological replicates (mice). p values compare biological replicates in Mann-Whitney test (B and D) and an unpaired t-test (F and H). ( I to K ) Calcium imaging of acutely purified DRG neurons from Ptges3-Flox or Ptges3-cKO mice. Images show fluorescent intensity of the calcium indicator (Fluo-4,AM) in DRG neurons acutely isolated (within 2 hours of purification) from Ptges3-Flox (top) or Ptges3-cKO (bottom) mice at baseline, during the addition of VTD and 20 seconds (s) after VTD addition (I). Representative neuron traces (K) and the maximum difference between the florescence after stimulation and during baseline (max delta F/F) (panel L) are shown. n = 3 distinct biological replicates (mice) per group, 8-26 cells per mouse. Mean ± SEM is shown for biological replicates. p values compare biological replicates in a Mann-Whitney test (B, D, and F) or an unpaired t-test (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
    Figure Legend Snippet: ( A to H ) RNAscope and immunostaining of NaV expression in vivo in Ptges3-Flox or Ptges3-cKO mice. Left panels: RNAscope images show expression of Na V 1.7 and Na V 1.8 transcripts in lumbar DRG neurons at P0 (A) and P28 (E) (see also , , and for E16 time point and supporting data). Number of Na V 1.7 and Na V 1.8 mRNAs per cell quantified using FishQuant from P0 (B) and P28 (D). n = 4-9 mice per group. Right panels: immunohistochemistry for Na V 1.7 and Na V 1.8 in lumbar DRG neurons at P0 (C) and P28 (G). Cellular fluorescence was quantified using Cellpose. Gray circles, mRNA count or fluorescence intensity in a DRG neuron; pink circles, the average of cells in each mouse. n = 2-5 mice per group. Mean ± SEM is shown for biological replicates (mice). p values compare biological replicates in Mann-Whitney test (B and D) and an unpaired t-test (F and H). ( I to K ) Calcium imaging of acutely purified DRG neurons from Ptges3-Flox or Ptges3-cKO mice. Images show fluorescent intensity of the calcium indicator (Fluo-4,AM) in DRG neurons acutely isolated (within 2 hours of purification) from Ptges3-Flox (top) or Ptges3-cKO (bottom) mice at baseline, during the addition of VTD and 20 seconds (s) after VTD addition (I). Representative neuron traces (K) and the maximum difference between the florescence after stimulation and during baseline (max delta F/F) (panel L) are shown. n = 3 distinct biological replicates (mice) per group, 8-26 cells per mouse. Mean ± SEM is shown for biological replicates. p values compare biological replicates in a Mann-Whitney test (B, D, and F) or an unpaired t-test (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Techniques Used: Immunostaining, Expressing, In Vivo, Immunohistochemistry, Fluorescence, MANN-WHITNEY, Imaging, Purification, Isolation

    ( A ) UMAP visualization of DRG scRNA-seq data in Ptges3-Flox mice at P4. ( B ) Cell identity composition heatmaps of CGRP and proprioceptor DRG neuron subtype populations. Dark red indicates high relative cell density. ( C ) Number of CGRP and proprioceptor DRG neurons are reduced by half in Ptges3-cKO mice. ( D to G ) RNAscope shows expression of Neun, Pvalb, or Calca , and Na V 1.8 transcripts in lumbar DRG neurons at P0 to label proprioceptor or CGRP neurons, respectively. Pvalb + or Calca + neuron numbers are normalized to Neun + cells to calculate the percentage of proprioceptor or CGRP DRG neurons respectively. n = 3-5 mice, p values compare biological replicates in an unpaired t-test. ( H and I ) Bar plots indicate expression of Na V 1.7 and Na V 1.8 in CGRP and proprioceptor DRG subpopulations, from scRNA-seq data. p values compare cells in an unpaired t-test. ( J and K ) Hot plate and Hargreaves tests indicate that paw withdrawal latencies in response to noxious heat was significantly lengthened in Ptges3-cKO mice. n = 14, 20 mice (hot plate); 32, 17 mice (Hargreaves). Control littermates in (K) were either Ptges3 fl/fl or Ptges3 fl/+ mice. ( L ) Pain response following 1% PFA injection into the hind paw indicated the typical biphasic response. The first phase (acute pain) was unaffected, but the second phase (inflammatory pain) was reduced by ~40% in Ptges3-cKO mice. n = 9-11 mice (also see  ). ( M ) Rotarod assay (32 RPM, constant speed) indicates decreased fall latency in Ptges3-cKO mice. n = 18, 17 mice. ( N ) Precise foot placement is severely impacted in Ptges3-cKO mice recorded in the horizontal ladder test with unevenly placed rungs. n = 16 mice for each group. p values compare biological replicates (triangles-females, circles-males) in an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
    Figure Legend Snippet: ( A ) UMAP visualization of DRG scRNA-seq data in Ptges3-Flox mice at P4. ( B ) Cell identity composition heatmaps of CGRP and proprioceptor DRG neuron subtype populations. Dark red indicates high relative cell density. ( C ) Number of CGRP and proprioceptor DRG neurons are reduced by half in Ptges3-cKO mice. ( D to G ) RNAscope shows expression of Neun, Pvalb, or Calca , and Na V 1.8 transcripts in lumbar DRG neurons at P0 to label proprioceptor or CGRP neurons, respectively. Pvalb + or Calca + neuron numbers are normalized to Neun + cells to calculate the percentage of proprioceptor or CGRP DRG neurons respectively. n = 3-5 mice, p values compare biological replicates in an unpaired t-test. ( H and I ) Bar plots indicate expression of Na V 1.7 and Na V 1.8 in CGRP and proprioceptor DRG subpopulations, from scRNA-seq data. p values compare cells in an unpaired t-test. ( J and K ) Hot plate and Hargreaves tests indicate that paw withdrawal latencies in response to noxious heat was significantly lengthened in Ptges3-cKO mice. n = 14, 20 mice (hot plate); 32, 17 mice (Hargreaves). Control littermates in (K) were either Ptges3 fl/fl or Ptges3 fl/+ mice. ( L ) Pain response following 1% PFA injection into the hind paw indicated the typical biphasic response. The first phase (acute pain) was unaffected, but the second phase (inflammatory pain) was reduced by ~40% in Ptges3-cKO mice. n = 9-11 mice (also see ). ( M ) Rotarod assay (32 RPM, constant speed) indicates decreased fall latency in Ptges3-cKO mice. n = 18, 17 mice. ( N ) Precise foot placement is severely impacted in Ptges3-cKO mice recorded in the horizontal ladder test with unevenly placed rungs. n = 16 mice for each group. p values compare biological replicates (triangles-females, circles-males) in an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Techniques Used: Expressing, Injection

    na v 1 8 antibody  (Alomone Labs)


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    Alomone Labs na v 1 8 antibody
    (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see  ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see  ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see  ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.
    Na V 1 8 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs"

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    Journal: bioRxiv

    doi: 10.1101/2022.10.04.510784

    (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see  ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see  ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see  ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.
    Figure Legend Snippet: (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Techniques Used:

    (A) Inhibiting Na v 1.8 with PF-24 (1 μM) did not affect spiking pattern (χ 2 =0.00, p=1.00, McNemar test) and modestly reduced firing rate (F 1,54 =9.745, p= 0.012, two-way RM ANOVA, n=10) in DIV4-7 neurons. (B) PF-24 did not affect rheobase (Z 12 =0.420, p=0.685, Wilcoxon Rank test) but did reduce spike height (T12=2.939, p=0.012, paired-t-test). *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A.
    Figure Legend Snippet: (A) Inhibiting Na v 1.8 with PF-24 (1 μM) did not affect spiking pattern (χ 2 =0.00, p=1.00, McNemar test) and modestly reduced firing rate (F 1,54 =9.745, p= 0.012, two-way RM ANOVA, n=10) in DIV4-7 neurons. (B) PF-24 did not affect rheobase (Z 12 =0.420, p=0.685, Wilcoxon Rank test) but did reduce spike height (T12=2.939, p=0.012, paired-t-test). *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A.

    Techniques Used:

    (A) Sample voltage clamp recordings show that sodium current was reduced by the Na V 1.7 inhibitor PF-71 (30 nM) and by the Na V 1.1/1.3 inhibitor ICA (1 μM). Peak current was significantly reduced by PF-71 and ICA (F 2,192 =26.361, p<0.001, two-way RM ANOVA; n=9). (B) PF-71 and ICA both significantly altered spiking pattern (χ 2 =4.17, p=0.041 and χ 2 =7.11, p=0.0077, respectively, McNemar tests) and significantly reduced firing rate (F 1.54 =40.659, p<0.001, n=10 and F 1.78 =35.156, p<0.001, n=14, respectively, two-way RM ANOVAs). (C) PF-71 significantly increased rheobase (Z 18 =3.464, p<0.001, Wilcoxon rank test) and decreased spike height (T 18 =7.946, p<0.001, paired t-test). ICA did not significantly alter rheobase (Z 18 =1.248, p=0.225) but did reduce spike height (T 18 =3.243, p=0.005). Neither drug affected resting membrane potential (T 15 =1.681, p=0.113 for PF-71; T 18 =-1.132, p=0.272 for ICA, paired t-test). The Na V 1.8 antagonist PF-24 had negligible effects at DIV4-7 (see  ). (D) A computational model reproduced the combined effects of Na V 1.3 and Na V 1.7 on spiking pattern (also see  ). PF-71 effect was simulated as a 70% reduction in Na V 1.7 . ICA effect was simulated as a 90% reduction in Na V 1.3 . *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and B.
    Figure Legend Snippet: (A) Sample voltage clamp recordings show that sodium current was reduced by the Na V 1.7 inhibitor PF-71 (30 nM) and by the Na V 1.1/1.3 inhibitor ICA (1 μM). Peak current was significantly reduced by PF-71 and ICA (F 2,192 =26.361, p<0.001, two-way RM ANOVA; n=9). (B) PF-71 and ICA both significantly altered spiking pattern (χ 2 =4.17, p=0.041 and χ 2 =7.11, p=0.0077, respectively, McNemar tests) and significantly reduced firing rate (F 1.54 =40.659, p<0.001, n=10 and F 1.78 =35.156, p<0.001, n=14, respectively, two-way RM ANOVAs). (C) PF-71 significantly increased rheobase (Z 18 =3.464, p<0.001, Wilcoxon rank test) and decreased spike height (T 18 =7.946, p<0.001, paired t-test). ICA did not significantly alter rheobase (Z 18 =1.248, p=0.225) but did reduce spike height (T 18 =3.243, p=0.005). Neither drug affected resting membrane potential (T 15 =1.681, p=0.113 for PF-71; T 18 =-1.132, p=0.272 for ICA, paired t-test). The Na V 1.8 antagonist PF-24 had negligible effects at DIV4-7 (see ). (D) A computational model reproduced the combined effects of Na V 1.3 and Na V 1.7 on spiking pattern (also see ). PF-71 effect was simulated as a 70% reduction in Na V 1.7 . ICA effect was simulated as a 90% reduction in Na V 1.3 . *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Techniques Used:

    (A) The computational model predicts that the Na V 1.8 conductance, which is “necessary” for repetitive spiking at DIV0 can, in principle, be replaced by Na V 1.7 (left), and vice versa at DIV4-7 (right). (B) Replacement experiments involved inhibiting native channels pharmacologically and then introducing virtual conductances using dynamic clamp. At DIV0 (left), inhibiting native Na V 1.8 (with PF-24) converted neurons to transient spiking, but introducing virtual Na V 1.7 reverted neurons to repetitive spiking (in 3 of 3 neurons tested). At DIV4-7, inhibiting native Na V 1.7 (with PF-71) converted the neuron to transient spiking, but introducing virtual Na V 1.8 reverted neurons to repetitive spiking (in 4 of 4 neurons tested). Parameters for virtual channels were identical to simulations except for the maximal conductance density, which was titrated in each cell. (C) Voltage (top) for first (left) and second (right) spikes in the DIV0 model aligned with voltage activation curves for each Na V isoform (bottom). Dashed line shows voltage threshold (defined as V where dV/dt reaches 5 mV/ms). (D) Conductance plotted against voltage to create a phase portrait (top) showing Na V conductance at different phases of the spike. Inset shows full voltage range; main graph zooms in on voltages near threshold. Bottom plots show current plotted over the same voltage range. For the first spike, Na V 1.7 (orange) mediates nearly all perithreshold inward current. For the second spike, voltage threshold is increased and Na V 1.8 (green) mediates nearly all perithreshold inward current because Na V 1.7 has inactivated (see  ). ( E-F ) In the DIV4-7 model, Na V 1.7 (orange) and Na V 1.3 (maroon) contribute to initiation of all spikes whereas the contribution of Na V 1.8 is negligible.
    Figure Legend Snippet: (A) The computational model predicts that the Na V 1.8 conductance, which is “necessary” for repetitive spiking at DIV0 can, in principle, be replaced by Na V 1.7 (left), and vice versa at DIV4-7 (right). (B) Replacement experiments involved inhibiting native channels pharmacologically and then introducing virtual conductances using dynamic clamp. At DIV0 (left), inhibiting native Na V 1.8 (with PF-24) converted neurons to transient spiking, but introducing virtual Na V 1.7 reverted neurons to repetitive spiking (in 3 of 3 neurons tested). At DIV4-7, inhibiting native Na V 1.7 (with PF-71) converted the neuron to transient spiking, but introducing virtual Na V 1.8 reverted neurons to repetitive spiking (in 4 of 4 neurons tested). Parameters for virtual channels were identical to simulations except for the maximal conductance density, which was titrated in each cell. (C) Voltage (top) for first (left) and second (right) spikes in the DIV0 model aligned with voltage activation curves for each Na V isoform (bottom). Dashed line shows voltage threshold (defined as V where dV/dt reaches 5 mV/ms). (D) Conductance plotted against voltage to create a phase portrait (top) showing Na V conductance at different phases of the spike. Inset shows full voltage range; main graph zooms in on voltages near threshold. Bottom plots show current plotted over the same voltage range. For the first spike, Na V 1.7 (orange) mediates nearly all perithreshold inward current. For the second spike, voltage threshold is increased and Na V 1.8 (green) mediates nearly all perithreshold inward current because Na V 1.7 has inactivated (see ). ( E-F ) In the DIV4-7 model, Na V 1.7 (orange) and Na V 1.3 (maroon) contribute to initiation of all spikes whereas the contribution of Na V 1.8 is negligible.

    Techniques Used: Activation Assay

    (A) Sample response at DIV0 showing that a virtual Na V 1.8 conductance applied with dynamic clamp restored repetitive spiking after inhibiting native Na V 1.8 channels with PF-24. This restoration was repeated in 3 of 3 neurons tested. (B) Sample recording at DIV4-7 showing that a virtual Na V 1.7 conductance restored repetitive spiking after inhibiting native Na V 1.7 channels with PF-71. This restoration was repeated in 4 of 4 neurons tested.
    Figure Legend Snippet: (A) Sample response at DIV0 showing that a virtual Na V 1.8 conductance applied with dynamic clamp restored repetitive spiking after inhibiting native Na V 1.8 channels with PF-24. This restoration was repeated in 3 of 3 neurons tested. (B) Sample recording at DIV4-7 showing that a virtual Na V 1.7 conductance restored repetitive spiking after inhibiting native Na V 1.7 channels with PF-71. This restoration was repeated in 4 of 4 neurons tested.

    Techniques Used:

    (A) In the DIV0 model, Na V 1.7 contributed to the first spike but its inactivation meant that all subsequent spikes relied exclusively on Na V 1.8. (B) In the DIV4-7 model, despite some inactivation of Na V 1.3 (red) and Na V 1.7 (green), the remaining current was still large enough to produce a net inward current sufficient to support repetitive spiking.
    Figure Legend Snippet: (A) In the DIV0 model, Na V 1.7 contributed to the first spike but its inactivation meant that all subsequent spikes relied exclusively on Na V 1.8. (B) In the DIV4-7 model, despite some inactivation of Na V 1.3 (red) and Na V 1.7 (green), the remaining current was still large enough to produce a net inward current sufficient to support repetitive spiking.

    Techniques Used:

    (A) Both Na V 1.8 and Na V 1.7 mRNA levels (relative to a housekeeping gene (HKG), see Methods) decreased significantly between DIV0 and DIV4-7 (factor 1: time, F 1,12 =56.677, p<0.001, factor 2: Na V isoform, F 1,12 =17.952, p=0.001, two-way ANOVA and Student-Newman-Keuls post-hoc tests on log transformed data, n=4 mice per time point) but more so for Na V 1.8 than for Na V 1.7 (interaction: time x isoform, F 1,12 = 11.455, p=0.005). The differential reduction translated into a significantly higher Na V 1.8: Na V 1.7 ratio on DIV0 than at DIV4-7 (T 6 =21.375, p<0.001, unpaired t-test). These changes may account for Na V 1.8 becoming unnecessary for repetitive spiking at DIV4-7 but cannot account for Na V 1.7 becoming necessary. (B) Immunoreactivity (IR) for Na V 1.8 protein exceeded Na V 1.7-IR at DIV0, but the opposite was true on DIV4-7. Na V -IR was measured relative to YFP-IR in the same cell, and then each cell’s Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the co-processed coverslip (left) or average Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the same animal (right). Both ratios were >1 at DIV0 but decreased significantly at DIV4-7 (U=78, p<0.001, n=37 for DIV0, n=40 for DIV4-7, Mann-Whitney test (left) and T 6 =4.046, p=0.007, unpaired t-test (right)). (C) Chronically applied cercosporamide (10 μM) mitigated the changes in Na V 1.8- and Na V 1.7-IR at DIV5 (Na V 1.8: H 3 =157.95, p<0.001; Na V 1.7: H 3 =80.662, p<0.001; One-way ANOVA on ranks, Dunn’s post-hoc tests, p<0.05 for all pairs). Panel on the right shows data normalized to baseline (DIV0) to emphasize relative changes.
    Figure Legend Snippet: (A) Both Na V 1.8 and Na V 1.7 mRNA levels (relative to a housekeeping gene (HKG), see Methods) decreased significantly between DIV0 and DIV4-7 (factor 1: time, F 1,12 =56.677, p<0.001, factor 2: Na V isoform, F 1,12 =17.952, p=0.001, two-way ANOVA and Student-Newman-Keuls post-hoc tests on log transformed data, n=4 mice per time point) but more so for Na V 1.8 than for Na V 1.7 (interaction: time x isoform, F 1,12 = 11.455, p=0.005). The differential reduction translated into a significantly higher Na V 1.8: Na V 1.7 ratio on DIV0 than at DIV4-7 (T 6 =21.375, p<0.001, unpaired t-test). These changes may account for Na V 1.8 becoming unnecessary for repetitive spiking at DIV4-7 but cannot account for Na V 1.7 becoming necessary. (B) Immunoreactivity (IR) for Na V 1.8 protein exceeded Na V 1.7-IR at DIV0, but the opposite was true on DIV4-7. Na V -IR was measured relative to YFP-IR in the same cell, and then each cell’s Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the co-processed coverslip (left) or average Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the same animal (right). Both ratios were >1 at DIV0 but decreased significantly at DIV4-7 (U=78, p<0.001, n=37 for DIV0, n=40 for DIV4-7, Mann-Whitney test (left) and T 6 =4.046, p=0.007, unpaired t-test (right)). (C) Chronically applied cercosporamide (10 μM) mitigated the changes in Na V 1.8- and Na V 1.7-IR at DIV5 (Na V 1.8: H 3 =157.95, p<0.001; Na V 1.7: H 3 =80.662, p<0.001; One-way ANOVA on ranks, Dunn’s post-hoc tests, p<0.05 for all pairs). Panel on the right shows data normalized to baseline (DIV0) to emphasize relative changes.

    Techniques Used: Transformation Assay, MANN-WHITNEY

    ( A ) Sample responses in DIV0 neurons from mice injected with CFA three days earlier. In 12 cells tested, PF-71 converted 5 neurons to transient spiking ( i ), encouraged repetitive spiking in 4 neurons ( ii ), and had no effect in 3 neurons ( iii ), thus highlighting increased heterogeneity after CFA. ( B ) At DIV0, the effect of PF-71 differed significantly between CFA and control neurons, converting 42% (5 of 12) CFA neurons from repetitive to transient spiking vs 0% (0 of 9) control neurons (p=0.0451, Fisher Exact test). Applying PF-24 to neurons that continued to spike repetitively after PF-71 had little effect on CFA neuron, converting only 13% (1 of 7) of CFA neurons vs 88% (7 of 8) of control neurons (p=0.001, Fisher Exact test). Together these results argue that Na V 1.7 contributes more and Na V 1.8 contributes less to nociceptor excitability after inflammation. (C) At DIV0, PF-71 significantly increased resting membrane potential (T 11 =-3.530, p=0.005, paired t-test) and rheobase (Z 11 =2.186, p=0.024, Wilcoxon rank test), and significantly decreased spike height (T 11 =4.413, p=0.001, paired t-test) in CFA neurons. Further addition of PF-24 significantly changed rheobase (Z 9 =2.176, p=0.023, Wilcoxon rank test) and action potential amplitude (T 9 =3.237, p=0.01, paired t-test) but did not affect resting membrane potential (T 9 =1.049, p=0.321, paired t-test). (D) Paw inflammation caused by CFA significantly altered thermal sensitivity (Hargreaves: F 5,65 =19.556, p<0.001, two-way RM ANOVA) and mechanical sensitivity (von Frey: F 4,52 =16.786, p<0.001). When given three days after CFA, PF-71 significantly reversed the altered sensitivities (Hargreaves: T 8 =-7.296, p<0.001; von Frey: T 8 =-4.341, p=0.002; paired t-tests) but had no effect in naive mice (Hargreaves: T 5 =-0.141, p=0.894; von Frey: T 5 =1.000, p=0.363). Insets show values for each animal before and 2 hours after PF-71 injection. *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests.
    Figure Legend Snippet: ( A ) Sample responses in DIV0 neurons from mice injected with CFA three days earlier. In 12 cells tested, PF-71 converted 5 neurons to transient spiking ( i ), encouraged repetitive spiking in 4 neurons ( ii ), and had no effect in 3 neurons ( iii ), thus highlighting increased heterogeneity after CFA. ( B ) At DIV0, the effect of PF-71 differed significantly between CFA and control neurons, converting 42% (5 of 12) CFA neurons from repetitive to transient spiking vs 0% (0 of 9) control neurons (p=0.0451, Fisher Exact test). Applying PF-24 to neurons that continued to spike repetitively after PF-71 had little effect on CFA neuron, converting only 13% (1 of 7) of CFA neurons vs 88% (7 of 8) of control neurons (p=0.001, Fisher Exact test). Together these results argue that Na V 1.7 contributes more and Na V 1.8 contributes less to nociceptor excitability after inflammation. (C) At DIV0, PF-71 significantly increased resting membrane potential (T 11 =-3.530, p=0.005, paired t-test) and rheobase (Z 11 =2.186, p=0.024, Wilcoxon rank test), and significantly decreased spike height (T 11 =4.413, p=0.001, paired t-test) in CFA neurons. Further addition of PF-24 significantly changed rheobase (Z 9 =2.176, p=0.023, Wilcoxon rank test) and action potential amplitude (T 9 =3.237, p=0.01, paired t-test) but did not affect resting membrane potential (T 9 =1.049, p=0.321, paired t-test). (D) Paw inflammation caused by CFA significantly altered thermal sensitivity (Hargreaves: F 5,65 =19.556, p<0.001, two-way RM ANOVA) and mechanical sensitivity (von Frey: F 4,52 =16.786, p<0.001). When given three days after CFA, PF-71 significantly reversed the altered sensitivities (Hargreaves: T 8 =-7.296, p<0.001; von Frey: T 8 =-4.341, p=0.002; paired t-tests) but had no effect in naive mice (Hargreaves: T 5 =-0.141, p=0.894; von Frey: T 5 =1.000, p=0.363). Insets show values for each animal before and 2 hours after PF-71 injection. *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests.

    Techniques Used: Injection

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    Alomone Labs anti nav1 8
    <t>Nav1.8</t> knockdown block rosacea-like development. A, Schematic diagram of intradermal injection of Nav1.8 for continuous 3 days before intradermal injection of LL37 in mice. B, The mRNA levels of Nav1.8 by siRNA in LL37-induced mice. C, The back skins of NC (negative control siRNA) and Nav1.8 knockdown (Nav1.8 siRNA) mice injected with LL37 or PBS. D, The reduced redness score, area of erythema and skin thickness of skin tissues in PBS/LL37-induced mice. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. E, HE staining of lesional skin of NC and Nav1.8 knockdown mice injected with LL37 or PBS. F, The mRNA levels of IL1β, IL-6, TLR2 and MMP-9 in mice skin lesion. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. G, The CD4 + T cells infiltration in mice skin lesion. H, The CD31 + microvascular in skin visualized by immunofluorescence. (n = 6 for each group).
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    1) Product Images from "Nav1.8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases"

    Article Title: Nav1.8 in keratinocytes contributes to ROS-mediated inflammation in inflammatory skin diseases

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102427

    Nav1.8 knockdown block rosacea-like development. A, Schematic diagram of intradermal injection of Nav1.8 for continuous 3 days before intradermal injection of LL37 in mice. B, The mRNA levels of Nav1.8 by siRNA in LL37-induced mice. C, The back skins of NC (negative control siRNA) and Nav1.8 knockdown (Nav1.8 siRNA) mice injected with LL37 or PBS. D, The reduced redness score, area of erythema and skin thickness of skin tissues in PBS/LL37-induced mice. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. E, HE staining of lesional skin of NC and Nav1.8 knockdown mice injected with LL37 or PBS. F, The mRNA levels of IL1β, IL-6, TLR2 and MMP-9 in mice skin lesion. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. G, The CD4 + T cells infiltration in mice skin lesion. H, The CD31 + microvascular in skin visualized by immunofluorescence. (n = 6 for each group).
    Figure Legend Snippet: Nav1.8 knockdown block rosacea-like development. A, Schematic diagram of intradermal injection of Nav1.8 for continuous 3 days before intradermal injection of LL37 in mice. B, The mRNA levels of Nav1.8 by siRNA in LL37-induced mice. C, The back skins of NC (negative control siRNA) and Nav1.8 knockdown (Nav1.8 siRNA) mice injected with LL37 or PBS. D, The reduced redness score, area of erythema and skin thickness of skin tissues in PBS/LL37-induced mice. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. E, HE staining of lesional skin of NC and Nav1.8 knockdown mice injected with LL37 or PBS. F, The mRNA levels of IL1β, IL-6, TLR2 and MMP-9 in mice skin lesion. Data represents the mean ± SEM. **p < 0.01, ***p < 0.001.2-way ANOVA test was used. G, The CD4 + T cells infiltration in mice skin lesion. H, The CD31 + microvascular in skin visualized by immunofluorescence. (n = 6 for each group).

    Techniques Used: Blocking Assay, Injection, Negative Control, Staining, Immunofluorescence

    Knockdown Nav1.8 attenuated psoriasis-like development in mice. A, Schematic diagram of NC/Nav1.8 siRNA-injected mice treated with IMQ or control. B, The mRNA levels of Nav1.8 silenced by siRNA in the skin tissues from IMQ-induced inflammation mice. Data represents the mean ± SEM. and *p < 0.05, **p < 0.01.2-way ANOVA test was used. C, The ears of NC and Nav1.8 knockdown mice treated with IMQ or control. D, The erythema score, scales score and ear thickness of psoriasis-induced inflammation mice. For statistical analysis, siRNA-IMQ group was compared with NC-IMQ group, *p < 0.05, **p < 0.01, ***p < 0.001.1-way ANOVA test was used. E, HE staining of lesional skin of WT and Nav1.8 knockdown mice treated with IMQ. F, The Ki67 expression visualized by immunofluorescence. G, The mRNA levels of IL1β, IL-6, IL17-A, IL-17F, IL-22, IL23A in IMQ-induced inflammation mice. H, The flow cytometry analysis revealed the immune cells infiltration in the skin lesion of IMQ-induced inflammation mice.
    Figure Legend Snippet: Knockdown Nav1.8 attenuated psoriasis-like development in mice. A, Schematic diagram of NC/Nav1.8 siRNA-injected mice treated with IMQ or control. B, The mRNA levels of Nav1.8 silenced by siRNA in the skin tissues from IMQ-induced inflammation mice. Data represents the mean ± SEM. and *p < 0.05, **p < 0.01.2-way ANOVA test was used. C, The ears of NC and Nav1.8 knockdown mice treated with IMQ or control. D, The erythema score, scales score and ear thickness of psoriasis-induced inflammation mice. For statistical analysis, siRNA-IMQ group was compared with NC-IMQ group, *p < 0.05, **p < 0.01, ***p < 0.001.1-way ANOVA test was used. E, HE staining of lesional skin of WT and Nav1.8 knockdown mice treated with IMQ. F, The Ki67 expression visualized by immunofluorescence. G, The mRNA levels of IL1β, IL-6, IL17-A, IL-17F, IL-22, IL23A in IMQ-induced inflammation mice. H, The flow cytometry analysis revealed the immune cells infiltration in the skin lesion of IMQ-induced inflammation mice.

    Techniques Used: Injection, Staining, Expressing, Immunofluorescence, Flow Cytometry

    List of primers used for Real-time PCR.
    Figure Legend Snippet: List of primers used for Real-time PCR.

    Techniques Used:

    Nav1.8 expression in rosacea and psoriasis. A, Immunohistochemistry (IHC) of Nav1.8 on skin tissues from HS (healthy individuals) and rosacea patients. Data represents the mean ± SEM. *P < 0.05, one-way ANOVA was used. B, IHC of Nav1.8 on skin tissues from HS and psoriasis patients. Data represents the mean ± SEM. *p < 0.05. Two-tailed unpaired Student's t-test was used. C, The immunofluorescence of Nav1.8 on skin tissues from PBS and LL37-induced mice. D, The immunofluorescence of Nav1.8 on skin tissues from control and IMQ-induced mice. E, qPCR analysis of Nav1.8 in skin tissues from PBS and LL37-induced mice. F, qPCR analysis of Nav1.8 in skin tissues from control and IMQ induced mice. n = 5 for each group, Data represents the mean ± SEM. ***p < 0.001. Two-tailed unpaired Student's t-test was used.
    Figure Legend Snippet: Nav1.8 expression in rosacea and psoriasis. A, Immunohistochemistry (IHC) of Nav1.8 on skin tissues from HS (healthy individuals) and rosacea patients. Data represents the mean ± SEM. *P < 0.05, one-way ANOVA was used. B, IHC of Nav1.8 on skin tissues from HS and psoriasis patients. Data represents the mean ± SEM. *p < 0.05. Two-tailed unpaired Student's t-test was used. C, The immunofluorescence of Nav1.8 on skin tissues from PBS and LL37-induced mice. D, The immunofluorescence of Nav1.8 on skin tissues from control and IMQ-induced mice. E, qPCR analysis of Nav1.8 in skin tissues from PBS and LL37-induced mice. F, qPCR analysis of Nav1.8 in skin tissues from control and IMQ induced mice. n = 5 for each group, Data represents the mean ± SEM. ***p < 0.001. Two-tailed unpaired Student's t-test was used.

    Techniques Used: Expressing, Immunohistochemistry, Two Tailed Test, Immunofluorescence

    Nav1.8 promoted the progression inflammatory skin disease by regulating cytokines expression in keratinocytes. A, The upregulated genes in NC_LL37 group compared to NC_PBS group. B, The downregulated genes in NC_LL37 group compared to NC_PBS group. C, The GO and KEGG enrichment analysis of the 2390 genes upregulated in NC_LL37 group which were attenuated by Nav1.8 siRNA. D, The T cell activation, Th1/Th17 differentiation, macrophage and neutrophils activation-related genes expression in NC_LL37 group compared to NC_PBS group and in Si_LL37 group compared to NC_LL37 group. E, PPI network analysis revealed the hub genes of these immune-cell related genes. F, The IL1β and IL6 expression in the keratinocytes of rosacea and psoriasis from HRA000809 and GSE166388 datasets. Data represents the mean ± SEM. *p < 0.05, **p < 0.01. Two-tailed unpaired Student's t-test was used. G, The IL1β and IL6 expression in skin lesion from NC and Nav1.8 knockdown mice injected with LL37 or PBS. H, The IL1β and IL6 expression in skin lesion from NC and Nav1.8 knockdown mice treated with IMQ or control visualized by immunofluorescence.
    Figure Legend Snippet: Nav1.8 promoted the progression inflammatory skin disease by regulating cytokines expression in keratinocytes. A, The upregulated genes in NC_LL37 group compared to NC_PBS group. B, The downregulated genes in NC_LL37 group compared to NC_PBS group. C, The GO and KEGG enrichment analysis of the 2390 genes upregulated in NC_LL37 group which were attenuated by Nav1.8 siRNA. D, The T cell activation, Th1/Th17 differentiation, macrophage and neutrophils activation-related genes expression in NC_LL37 group compared to NC_PBS group and in Si_LL37 group compared to NC_LL37 group. E, PPI network analysis revealed the hub genes of these immune-cell related genes. F, The IL1β and IL6 expression in the keratinocytes of rosacea and psoriasis from HRA000809 and GSE166388 datasets. Data represents the mean ± SEM. *p < 0.05, **p < 0.01. Two-tailed unpaired Student's t-test was used. G, The IL1β and IL6 expression in skin lesion from NC and Nav1.8 knockdown mice injected with LL37 or PBS. H, The IL1β and IL6 expression in skin lesion from NC and Nav1.8 knockdown mice treated with IMQ or control visualized by immunofluorescence.

    Techniques Used: Expressing, Activation Assay, Two Tailed Test, Injection, Immunofluorescence

    Nav1.8 induced ROS accumulation in keratinocyte by interacting with SOD2 directly. A, Nav.18 expression in NC and Nav1.8 knockdown keratinocyte treated with TNFα. B, IL-1β and IL-6 expression in NC and Nav1.8 knockdown keratinocyte treated with TNFα. C, P38, p-P38, P65, p-P65 expression in control and Nav1.8 knockdown keratinocyte treated with TNFα. D, IL-1β and IL-6 expression in control and A-803467-treated keratinocyte stimulated with TNFα. E, The ROS levels of keratinocyte treated with TNFα and Nav1.8 siRNA or A803467. F, The DHE straining of skin lesion from NC and Nav1.8 knockdown mice treated with IMQ or control or PBS or LL37. G, GO enrichment analysis of potential interaction proteins of Nav1.8 C-terminal. H, The ROS levels of and mRNA levels of IL-1β and IL-6 in Nav1.8-C overexpressed keratinocyte treated with mitoTEMPO. I, Co-IP revealed the interaction between Nav1.8-C and SOD2 in keratinocytes. J, The endogenous Co-IP revealed the interaction between Nav1.8 and SOD2 in DRG (Dorsal root ganglion). Data represents the mean ± SEM. and *p < 0.05, **p < 0.01, ***p < 0.001.2-way ANOVA test was used.
    Figure Legend Snippet: Nav1.8 induced ROS accumulation in keratinocyte by interacting with SOD2 directly. A, Nav.18 expression in NC and Nav1.8 knockdown keratinocyte treated with TNFα. B, IL-1β and IL-6 expression in NC and Nav1.8 knockdown keratinocyte treated with TNFα. C, P38, p-P38, P65, p-P65 expression in control and Nav1.8 knockdown keratinocyte treated with TNFα. D, IL-1β and IL-6 expression in control and A-803467-treated keratinocyte stimulated with TNFα. E, The ROS levels of keratinocyte treated with TNFα and Nav1.8 siRNA or A803467. F, The DHE straining of skin lesion from NC and Nav1.8 knockdown mice treated with IMQ or control or PBS or LL37. G, GO enrichment analysis of potential interaction proteins of Nav1.8 C-terminal. H, The ROS levels of and mRNA levels of IL-1β and IL-6 in Nav1.8-C overexpressed keratinocyte treated with mitoTEMPO. I, Co-IP revealed the interaction between Nav1.8-C and SOD2 in keratinocytes. J, The endogenous Co-IP revealed the interaction between Nav1.8 and SOD2 in DRG (Dorsal root ganglion). Data represents the mean ± SEM. and *p < 0.05, **p < 0.01, ***p < 0.001.2-way ANOVA test was used.

    Techniques Used: Expressing, Co-Immunoprecipitation Assay

    Nav1.8 affects the translocation and acetylation levels of SOD2. A, SOD2 expression in NC and Nav1.8 knockdown keratinocytes treated with TNFα. B, SOD2 activity of NC and Nav1.8 knockdown keratinocyte treated with TNFα, or SOD2 activity of Nav1.8C-overexpressed keratinocyte cells, or SOD2 activity of A803467 treated keratinocyte cells. C, Western blots revealed the effects of Nav1.8-siRNA, Nav1.8-C, or TNFα on the levels of SOD2 in the cytosolic and mitochondrial fractions. D, The immunofluorescence revealed the mitochondrial SOD2 localization. E, Western blots revealed the effects of Nav1.8-siRNA, Nav1.8-C, or TNFα on the levels of SOD2 K68 acetylation.
    Figure Legend Snippet: Nav1.8 affects the translocation and acetylation levels of SOD2. A, SOD2 expression in NC and Nav1.8 knockdown keratinocytes treated with TNFα. B, SOD2 activity of NC and Nav1.8 knockdown keratinocyte treated with TNFα, or SOD2 activity of Nav1.8C-overexpressed keratinocyte cells, or SOD2 activity of A803467 treated keratinocyte cells. C, Western blots revealed the effects of Nav1.8-siRNA, Nav1.8-C, or TNFα on the levels of SOD2 in the cytosolic and mitochondrial fractions. D, The immunofluorescence revealed the mitochondrial SOD2 localization. E, Western blots revealed the effects of Nav1.8-siRNA, Nav1.8-C, or TNFα on the levels of SOD2 K68 acetylation.

    Techniques Used: Translocation Assay, Expressing, Activity Assay, Western Blot, Immunofluorescence

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    Alomone Labs goat anti nav1 8
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Goat Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti nav1 8
    Antibodies used for western blotting and fluorescent immunohistochemistry.
    Anti Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs scn10 a
    Antibodies used for western blotting and fluorescent immunohistochemistry.
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    Alomone Labs rabbit anti rat nav1 8
    (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of <t>Nav1.8-</t> and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.
    Rabbit Anti Rat Nav1 8, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs nav1 8 protein
    Protein expressions of <t>Nav1.3,</t> <t>Nav1.8,</t> and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.
    Nav1 8 Protein, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs nav1 8
    <t>Nav1.7</t> and <t>Nav1.8</t> expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.
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    Alomone Labs na v 1 8
    ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in  or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.
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    Alomone Labs na v 1 8 antibody
    (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see  ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see  ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see  ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.
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    Image Search Results


    Antibodies used for western blotting and fluorescent immunohistochemistry.

    Journal: Molecular Pain

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    doi: 10.1177/17448069211069255

    Figure Lengend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Article Snippet: Goat anti-Nav1.8 , Alomone , SCN10 A , Donkey anti-goat 647 , Sodium channel 1.8; nociceptors.

    Techniques: Western Blot, Immunohistochemistry

    Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Journal: Molecular Pain

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    doi: 10.1177/17448069211069255

    Figure Lengend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Article Snippet: Goat anti-Nav1.8 , Alomone , SCN10 A , Donkey anti-goat 647 , Sodium channel 1.8; nociceptors.

    Techniques:

    Antibodies used for western blotting and fluorescent immunohistochemistry.

    Journal: Molecular Pain

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    doi: 10.1177/17448069211069255

    Figure Lengend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Article Snippet: The tissue sections were then washed with 0.1 m PBS three times and then incubated with the corresponding fluorescent-conjugated secondary antibody goat anti-rabbit Alexa-488 or 568 (1:300; Molecular Probes, Eugen, OR, USA), goat anti-mouse Alexa-488 or 568 (1:300; Molecular Probes), or donkey anti-goat Alexa-647 (1:300; Molecular Probes) for 90 min, rinsed, then subjected to a second round of primary immune-labeling overnight at room temperature with primary antibody for Isolectin IB4 (1:500; Fischer Scientific, I21414) in 0.1 m m CaCl 2 , rabbit anti-TRPV1 (1:1000; Alomone, ACC-030), goat anti-Nav1.8 (1:1000; Alomone, SCN10 A), goat anti-CGRP (1:1000; Abcam, AB36001) or rabbit anti-CGRP (1:1000; Immunostar 24,112), guinea pig anti-PGP9.5 (1:1000; Millipore, AB5898), or chicken anti-NF200 (1:1000; Abcam, AB4680).

    Techniques: Western Blot, Immunohistochemistry

    Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Journal: Molecular Pain

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    doi: 10.1177/17448069211069255

    Figure Lengend Snippet: Sigma-1 receptors and progesterone metabolizing enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are highly expressed across various neuron populations in female rat trigeminal ganglia neurons. Quantification of the percentage of Sigma-1 receptors (Sig-1R), 5α-reductase, or 3α-hydroxysteroid dehydrogenase (3α-HSD) that are co-localized within the following neuron populations: protein gene product 9.5 (PGP9.5; neurons and nerve fibers; A), neurofilament heavy (NF200; myelinated neurons; B), sodium channel 1.8 (Nav1.8; nociceptors; C), isolectin IB4 (IB4, non-peptidergic neurons; D), transient receptor potential vanilloid 1 (TRPV1; nociceptor subpopulation; E), or calcitonin gene-related peptide (CGRP; peptidergic neurons; F).

    Article Snippet: The tissue sections were then washed with 0.1 m PBS three times and then incubated with the corresponding fluorescent-conjugated secondary antibody goat anti-rabbit Alexa-488 or 568 (1:300; Molecular Probes, Eugen, OR, USA), goat anti-mouse Alexa-488 or 568 (1:300; Molecular Probes), or donkey anti-goat Alexa-647 (1:300; Molecular Probes) for 90 min, rinsed, then subjected to a second round of primary immune-labeling overnight at room temperature with primary antibody for Isolectin IB4 (1:500; Fischer Scientific, I21414) in 0.1 m m CaCl 2 , rabbit anti-TRPV1 (1:1000; Alomone, ACC-030), goat anti-Nav1.8 (1:1000; Alomone, SCN10 A), goat anti-CGRP (1:1000; Abcam, AB36001) or rabbit anti-CGRP (1:1000; Immunostar 24,112), guinea pig anti-PGP9.5 (1:1000; Millipore, AB5898), or chicken anti-NF200 (1:1000; Abcam, AB4680).

    Techniques:

    Antibodies used for western blotting and fluorescent immunohistochemistry.

    Journal: Molecular Pain

    Article Title: Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone

    doi: 10.1177/17448069211069255

    Figure Lengend Snippet: Antibodies used for western blotting and fluorescent immunohistochemistry.

    Article Snippet: Goat anti-Nav1.8 , Alomone , SCN10 A , Donkey anti-goat 647 , Sodium channel 1.8; nociceptors.

    Techniques: Western Blot, Immunohistochemistry

    (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of Nav1.8- and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.

    Journal: PLoS ONE

    Article Title: Antisense-Mediated Knockdown of Na V 1.8, but Not Na V 1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

    doi: 10.1371/journal.pone.0019865

    Figure Lengend Snippet: (A) Representative RT-PCR results of five isoforms of voltage-gated sodium channels. Amplicons of Na V 1.1, Na V 1.6, Na V 1.7, Na V 1.8, Na V 1.9 and ß-actin were 540 bp, 509 bp, 441 bp, 515 bp, 572 bp and 229 bp, respectively. (B) Averaged fold changes of mRNA expression as normalized with naive control (n = 3). (C) Double immunofluorescent labeling of DRG neurons by anti-Na V 1.8 (red) and anti-NF-200 (green) or anti-Na V 1.9 (red) and anti-NF-200 (green) antibodies. (D) The percentage of Nav1.8- and Nav1.9-positive profiles as a proportion of the total number of DRG neurons before and after CFA treatment (n = 8). (E) Western blotting examples of Na V 1.8 and Na V 1.9 in naive and CFA-treated DRG neurons. (F) Averaged protein expression of Na V 1.8 and Na V 1.9 between naive and CFA-treated DRGs (normalized with the internal control tubulin) (n = 3 for each group). CFA, complete Freund's adjuvant. * p<0.05, **p<0.01, ***p<0.001.

    Article Snippet: The primary antibodies were mouse anti-Neurofilament 200 monoclonal antibody (NF-200, 1∶200, Sigma, USA), rabbit anti-rat Nav1.8 and Nav1.9 antibodies (1∶200, Alomone, Israel).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Labeling, Western Blot

    Protein expressions of Nav1.3, Nav1.8, and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.

    Journal: BMC Neurology

    Article Title: Pretreatment with intrathecal amitriptyline potentiates anti-hyperalgesic effects of post-injury intra-peritoneal amitriptyline following spinal nerve ligation

    doi: 10.1186/1471-2377-12-44

    Figure Lengend Snippet: Protein expressions of Nav1.3, Nav1.8, and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks ( A ). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively ( B ). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 ( C ) . Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). * p < 0.05; ** p < 0.01; ☆: p < 0.001,NS: not significant. POD: postoperative day; S: S ham; L: L igation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally ( A bdomen) post-injury; P: P retreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.

    Article Snippet: The expressions of Nav1.3, Nav1.7 and Nav1.8 protein were detected using rabbit primary antibodies (Alomone Labs, Jerusalem, Israel) followed by reaction with horseradish peroxidase-conjugated mouse anti-rabbit antibody (Santa Cruz, Biotechnology, Santa Cruz, CA).

    Techniques: Western Blot, Inhibition, Ligation

    Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

    doi: 10.1155/2013/312184

    Figure Lengend Snippet: Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar carrageenan injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the carrageenan-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) Carrageenan-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the carrageenan-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Article Snippet: DRGs were incubated with primary antibodies prepared in blocking solution at 4°C overnight against Nav1.7 (1 : 1000, Alomone), Nav1.8 (1 : 1000, Alomone), and Nav1.9 (1 : 1000, Alomone).

    Techniques: Injection

    Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the CFA-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) CFA-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the CFA-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

    doi: 10.1155/2013/312184

    Figure Lengend Snippet: Nav1.7 and Nav1.8 expressions were increased in ipsilateral DRGs after intraplantar CFA injection and further attenuated by EA at the ST36 acupoint in mice, though Nav1.9 was not different. (a)–(c) Nav1.7, Nav1.8, and Nav1.9 immunoreactive neurons were found in lumbar DRGs at the ipsilateral site of the saline-injected group. (d)-(e) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the CFA-injected group, but (f) Nav1.9 immunoreactive neurons were not increased. (g)-(h) CFA-induced increases of Nav1.7 and Nav1.8 were attenuated by EA, as compared to those of the CFA-induced group. (i) Nav1.9 immunoreactive neurons were not altered by EA at the ipsilateral site of inflammation. (j)-(k) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-Acu group. (l) Nav1.9 immunoreactive neurons were not altered in the S-Acu group. (m)-(n) Nav1.7 and Nav1.8 immunoreactive neurons were increased in the S-GM group. (o) Nav1.9 immunoreactive neurons were not altered in the S-GM group. Scale bar = 50 um.

    Article Snippet: DRGs were incubated with primary antibodies prepared in blocking solution at 4°C overnight against Nav1.7 (1 : 1000, Alomone), Nav1.8 (1 : 1000, Alomone), and Nav1.9 (1 : 1000, Alomone).

    Techniques: Injection

    Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan- and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan- and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan- and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

    doi: 10.1155/2013/312184

    Figure Lengend Snippet: Nav1.7 and Nav1.8 protein levels were increased in lumbar DRGs in both intraplantar carrageenan- and CFA-induced inflammation and further attenuated by EA at the ST36 acupoint in mice, but Nav1.9 proteins were not altered. (a) DRGs lysates were immunoreactive with specific antibodies to Nav1.7 and a substantially increased signal at the ipsilateral site, as compared to that of the saline-injected group. Nav1.7 protein levels were attenuated by EA at the ST36 acupoint, as compared to that of the carrageenan- and CFA-induced groups. (b) Nav1.8 displayed similar results to Nav1.7. The protein levels of S-Acu and S-GM were similar to inflamed but not EA group. (c) Nav1.9 protein levels were not changed in both the carrageenan- and CFA-injected sites. Nav1.9 protein levels were not attenuated by EA at the ST36 acupoint, as compared to those of the carrageenan- and CFA-induced groups, either. Nav1.9 proteins were not altered at the ipsilateral site of inflammation and EA stimulation.

    Article Snippet: DRGs were incubated with primary antibodies prepared in blocking solution at 4°C overnight against Nav1.7 (1 : 1000, Alomone), Nav1.8 (1 : 1000, Alomone), and Nav1.9 (1 : 1000, Alomone).

    Techniques: Injection

    Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3–L5 DRGs in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups. The percentage of Nav protein levels from lumbar DRGs was presented in each group. * P < 0.05, as compared to control group. # P < 0.05; comparison between inflammation and EA groups.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Reduces Carrageenan- and CFA-Induced Inflammatory Pain Accompanied by Changing the Expression of Nav1.7 and Nav1.8, rather than Nav1.9, in Mice Dorsal Root Ganglia

    doi: 10.1155/2013/312184

    Figure Lengend Snippet: Protein levels of Nav1.7, Nav1.8, and Nav1.9 in the L3–L5 DRGs in mice in control, Car, EA, S-Acu, S-GM, CFA, EA, S-Acu, S-GM groups. The percentage of Nav protein levels from lumbar DRGs was presented in each group. * P < 0.05, as compared to control group. # P < 0.05; comparison between inflammation and EA groups.

    Article Snippet: DRGs were incubated with primary antibodies prepared in blocking solution at 4°C overnight against Nav1.7 (1 : 1000, Alomone), Nav1.8 (1 : 1000, Alomone), and Nav1.9 (1 : 1000, Alomone).

    Techniques:

    ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in  or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( Caption continued on next page .) ( A ) Images show Neurofilament and DAPI staining in immunopanned DRG neurons as described in or unpanned DRG neurons plated after dissociation without the panning steps. Even with the use of the mitotic inhibitor FUDR, unpanned DRG neuron cultures had significant contamination with non-neural cells, negatively stained for Neurofilament. ( B ) Untreated and SCCM treated DRG neurons exhibited statistically similar resting membrane potentials. n = 29 total cells from 5–7 distinct biological replicates per treatment group. ( C ) Bright field images indicate that DRG neuronal health is unaffected by Act-D treatment, consistent with prior studies. ( D ) The addition of Schwann cell growth media to DRG neurons was insufficient to increase expression of Na V 1.7 and Na V 1.8 transcripts, suggesting the effects of SCCM are specific to a Schwann cell-secreted molecule(s). Gray circles, mRNA number of an individual DRG neuron for the indicated genes; colored circles, the average # of mRNAs per cell in each biological replicate. Mean ± SEM is shown for the biological replicates (not significantly different in a paired t-test). n = 2 distinct biological replicates per group. ( E ) Immunopanned DRG neurons showed normal cell growth and neurofilament staining, all unaffected by SCCM treatment.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Staining, Expressing

    ( Caption continued on next page .) ( A ) DESI-MS analysis of SCCM. The expected m/z of PGE 2 is 351.21770 and a m/z of 351.21824 ± 1.6 ppm was detected in the SCCM sample. ( B) SCCM and PGE 2 treatments enhanced Na V 1.6, Na v 1.7 and Na v 1.8 protein levels in DRG neurons. n = 80 to 328 cells from 1-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. Untreated condition is replotted from  , as these experiments were done concurrently. ( C ) SCCM and PGE 2 treatments did not cause a major change in protein expression of indicated genes, except a significant change in Na v 1.5 protein levels. Fluorescence levels were quantified using cellpose. Gray circles, fluorescent intensity of an individual DRG neuron for the indicated genes. n = 70 to 331 cells from 2-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. p values compare cells in a one-way ANOVA and Tukey test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( Caption continued on next page .) ( A ) DESI-MS analysis of SCCM. The expected m/z of PGE 2 is 351.21770 and a m/z of 351.21824 ± 1.6 ppm was detected in the SCCM sample. ( B) SCCM and PGE 2 treatments enhanced Na V 1.6, Na v 1.7 and Na v 1.8 protein levels in DRG neurons. n = 80 to 328 cells from 1-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. Untreated condition is replotted from , as these experiments were done concurrently. ( C ) SCCM and PGE 2 treatments did not cause a major change in protein expression of indicated genes, except a significant change in Na v 1.5 protein levels. Fluorescence levels were quantified using cellpose. Gray circles, fluorescent intensity of an individual DRG neuron for the indicated genes. n = 70 to 331 cells from 2-4 distinct biological replicates per treatment group. Mean ± SEM is shown for cells. p values compare cells in a one-way ANOVA and Tukey test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Expressing, Fluorescence

    ( A ) Cartoon depicts size fractionation and mass spectrometry steps that identified PGE 2 as the excitability-inducing molecule in SCCM. ( B ) PGE 2 treatment (1 μM, 16-28 hr) increased Na V expression in DRG neurons, similar to SCCM. n = 6 distinct biological replicates per treatment group. Bottom images: Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1-EP4) antagonists blocked the SCCM-induced transcriptional increase in Na V s (see also representative images for DMSO-treated DRG neurons in  ). n = 4-6 distinct biological replicates per treatment group. ( C) PGE 2 treatment enhanced Na V protein levels in DRG neurons. Images show immunohistochemistry for indicated genes in single representative DRG neurons that were either untreated (top) or treated with PGE 2 overnight (bottom) (see also quantification in  ). ( D and E ) Quantification of the RNAscope results shown in (B), using FishQuant. Controls were either untreated cells or cells treated with DMSO vehicle (solvent for PGE 2 ). Mean ± SEM is shown for biological replicates. p values compare biological replicates in a paired t-test (D) or mixed-effect analysis (E). ( F and G ) DRG neurons treated with PGE 2 (1 μM, 16-28 hr) fired significantly more action potentials at suprathreshold current injections and exhibited a decrease in the firing threshold, similar to SCCM. Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1–EP4) antagonists blocked the excitability-inducing effect of SCCM. DRG neurons treated with 1 μM PGD 2 , a constitutional isomer of PGE 2 , remained hypoexcitable (see also  ). Mean ± SEM is shown for cells, p values compare cells in a one-way ANOVA and Tukey test. n = 20-29 total cells from 3–7 biological replicates. Untreated and SCCM conditions are replotted from  and  , as these experiments were done concurrently. ( H-I ) Injection of dmPGE 2 into the P0 sciatic nerve increased Na V 1.7 and Na V 1.8 transcript levels in lumbar DRG neurons compared to controls (the third fluorescence channel was reserved for Neun ; see  ). Results were quantified using FishQuant (I). Gray circles, Na V mRNA count in a single DRG neuron; colored circles, the average of cells in each biological replicate. Mean ± SEM is shown for biological replicates. p values compare biological replicates in cells in a one-way ANOVA and Tukey test. n = 3 mice per treatment group (at least 45 DRG neurons per mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( A ) Cartoon depicts size fractionation and mass spectrometry steps that identified PGE 2 as the excitability-inducing molecule in SCCM. ( B ) PGE 2 treatment (1 μM, 16-28 hr) increased Na V expression in DRG neurons, similar to SCCM. n = 6 distinct biological replicates per treatment group. Bottom images: Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1-EP4) antagonists blocked the SCCM-induced transcriptional increase in Na V s (see also representative images for DMSO-treated DRG neurons in ). n = 4-6 distinct biological replicates per treatment group. ( C) PGE 2 treatment enhanced Na V protein levels in DRG neurons. Images show immunohistochemistry for indicated genes in single representative DRG neurons that were either untreated (top) or treated with PGE 2 overnight (bottom) (see also quantification in ). ( D and E ) Quantification of the RNAscope results shown in (B), using FishQuant. Controls were either untreated cells or cells treated with DMSO vehicle (solvent for PGE 2 ). Mean ± SEM is shown for biological replicates. p values compare biological replicates in a paired t-test (D) or mixed-effect analysis (E). ( F and G ) DRG neurons treated with PGE 2 (1 μM, 16-28 hr) fired significantly more action potentials at suprathreshold current injections and exhibited a decrease in the firing threshold, similar to SCCM. Incubation with neutralizing PGE 2 antibody or PGE 2 receptor (EP1–EP4) antagonists blocked the excitability-inducing effect of SCCM. DRG neurons treated with 1 μM PGD 2 , a constitutional isomer of PGE 2 , remained hypoexcitable (see also ). Mean ± SEM is shown for cells, p values compare cells in a one-way ANOVA and Tukey test. n = 20-29 total cells from 3–7 biological replicates. Untreated and SCCM conditions are replotted from and , as these experiments were done concurrently. ( H-I ) Injection of dmPGE 2 into the P0 sciatic nerve increased Na V 1.7 and Na V 1.8 transcript levels in lumbar DRG neurons compared to controls (the third fluorescence channel was reserved for Neun ; see ). Results were quantified using FishQuant (I). Gray circles, Na V mRNA count in a single DRG neuron; colored circles, the average of cells in each biological replicate. Mean ± SEM is shown for biological replicates. p values compare biological replicates in cells in a one-way ANOVA and Tukey test. n = 3 mice per treatment group (at least 45 DRG neurons per mouse). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Fractionation, Mass Spectrometry, Expressing, Incubation, Immunohistochemistry, Injection, Fluorescence

    ( Caption continued on next page .) ( A to D ) RNAscope shows expression of Na V 1.6 , Na V 1.7 and Na V 1.8 transcripts in DRG neurons treated with SCCM from Flox ( Ptges3 fl/fl ) or cKO ( Ptges3 fl/fl ;Dhh CRE/+ ) mice. Micrographs of SCCM treatment from cHET ( Ptges3 fl/+ ;Dhh CRE/+ ), Dhh CRE/+ and WT mice are not shown but are also quantified in ( E to G ). ( E to G ) Conditioned media from Ptges3 conditional knockout Schwann cells (cKO) failed to enhance neuronal Na V 1.8 expression, contrary to Schwann cells collected from controls (Flox, Dhh CRE/+ , or WT). We detected a slight but significant change in Na V 1.6 and Na V 1.7 expression in DRG neurons following the addition of cKO media; however, this effect was lower than the fold increase observed with WT, Flox, or Dhh CRE/+ SCCM addition. The addition of PGE 2 to cKO media rescued the loss of Na v expression-inducing effect in cKO SCCM. Gray circles, mRNA number of an individual DRG neuron for the indicated genes. Mean ± SEM of all cells; p values compare cells in a one-way ANOVA and Tukey test. Significant increases in comparison to untreated DRG neurons or between groups indicated by brackets are shown. n = 61–195 DRG neurons from 2–4 biological replicates per group. p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( Caption continued on next page .) ( A to D ) RNAscope shows expression of Na V 1.6 , Na V 1.7 and Na V 1.8 transcripts in DRG neurons treated with SCCM from Flox ( Ptges3 fl/fl ) or cKO ( Ptges3 fl/fl ;Dhh CRE/+ ) mice. Micrographs of SCCM treatment from cHET ( Ptges3 fl/+ ;Dhh CRE/+ ), Dhh CRE/+ and WT mice are not shown but are also quantified in ( E to G ). ( E to G ) Conditioned media from Ptges3 conditional knockout Schwann cells (cKO) failed to enhance neuronal Na V 1.8 expression, contrary to Schwann cells collected from controls (Flox, Dhh CRE/+ , or WT). We detected a slight but significant change in Na V 1.6 and Na V 1.7 expression in DRG neurons following the addition of cKO media; however, this effect was lower than the fold increase observed with WT, Flox, or Dhh CRE/+ SCCM addition. The addition of PGE 2 to cKO media rescued the loss of Na v expression-inducing effect in cKO SCCM. Gray circles, mRNA number of an individual DRG neuron for the indicated genes. Mean ± SEM of all cells; p values compare cells in a one-way ANOVA and Tukey test. Significant increases in comparison to untreated DRG neurons or between groups indicated by brackets are shown. n = 61–195 DRG neurons from 2–4 biological replicates per group. p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Expressing, Knock-Out

    ( A to H ) RNAscope and immunostaining of NaV expression in vivo in Ptges3-Flox or Ptges3-cKO mice. Left panels: RNAscope images show expression of Na V 1.7 and Na V 1.8 transcripts in lumbar DRG neurons at P0 (A) and P28 (E) (see also  ,  , and  for E16 time point and supporting data). Number of Na V 1.7 and Na V 1.8 mRNAs per cell quantified using FishQuant from P0 (B) and P28 (D). n = 4-9 mice per group. Right panels: immunohistochemistry for Na V 1.7 and Na V 1.8 in lumbar DRG neurons at P0 (C) and P28 (G). Cellular fluorescence was quantified using Cellpose. Gray circles, mRNA count or fluorescence intensity in a DRG neuron; pink circles, the average of cells in each mouse. n = 2-5 mice per group. Mean ± SEM is shown for biological replicates (mice). p values compare biological replicates in Mann-Whitney test (B and D) and an unpaired t-test (F and H). ( I to K ) Calcium imaging of acutely purified DRG neurons from Ptges3-Flox or Ptges3-cKO mice. Images show fluorescent intensity of the calcium indicator (Fluo-4,AM) in DRG neurons acutely isolated (within 2 hours of purification) from Ptges3-Flox (top) or Ptges3-cKO (bottom) mice at baseline, during the addition of VTD and 20 seconds (s) after VTD addition (I). Representative neuron traces (K) and the maximum difference between the florescence after stimulation and during baseline (max delta F/F) (panel L) are shown. n = 3 distinct biological replicates (mice) per group, 8-26 cells per mouse. Mean ± SEM is shown for biological replicates. p values compare biological replicates in a Mann-Whitney test (B, D, and F) or an unpaired t-test (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( A to H ) RNAscope and immunostaining of NaV expression in vivo in Ptges3-Flox or Ptges3-cKO mice. Left panels: RNAscope images show expression of Na V 1.7 and Na V 1.8 transcripts in lumbar DRG neurons at P0 (A) and P28 (E) (see also , , and for E16 time point and supporting data). Number of Na V 1.7 and Na V 1.8 mRNAs per cell quantified using FishQuant from P0 (B) and P28 (D). n = 4-9 mice per group. Right panels: immunohistochemistry for Na V 1.7 and Na V 1.8 in lumbar DRG neurons at P0 (C) and P28 (G). Cellular fluorescence was quantified using Cellpose. Gray circles, mRNA count or fluorescence intensity in a DRG neuron; pink circles, the average of cells in each mouse. n = 2-5 mice per group. Mean ± SEM is shown for biological replicates (mice). p values compare biological replicates in Mann-Whitney test (B and D) and an unpaired t-test (F and H). ( I to K ) Calcium imaging of acutely purified DRG neurons from Ptges3-Flox or Ptges3-cKO mice. Images show fluorescent intensity of the calcium indicator (Fluo-4,AM) in DRG neurons acutely isolated (within 2 hours of purification) from Ptges3-Flox (top) or Ptges3-cKO (bottom) mice at baseline, during the addition of VTD and 20 seconds (s) after VTD addition (I). Representative neuron traces (K) and the maximum difference between the florescence after stimulation and during baseline (max delta F/F) (panel L) are shown. n = 3 distinct biological replicates (mice) per group, 8-26 cells per mouse. Mean ± SEM is shown for biological replicates. p values compare biological replicates in a Mann-Whitney test (B, D, and F) or an unpaired t-test (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Immunostaining, Expressing, In Vivo, Immunohistochemistry, Fluorescence, MANN-WHITNEY, Imaging, Purification, Isolation

    ( A ) UMAP visualization of DRG scRNA-seq data in Ptges3-Flox mice at P4. ( B ) Cell identity composition heatmaps of CGRP and proprioceptor DRG neuron subtype populations. Dark red indicates high relative cell density. ( C ) Number of CGRP and proprioceptor DRG neurons are reduced by half in Ptges3-cKO mice. ( D to G ) RNAscope shows expression of Neun, Pvalb, or Calca , and Na V 1.8 transcripts in lumbar DRG neurons at P0 to label proprioceptor or CGRP neurons, respectively. Pvalb + or Calca + neuron numbers are normalized to Neun + cells to calculate the percentage of proprioceptor or CGRP DRG neurons respectively. n = 3-5 mice, p values compare biological replicates in an unpaired t-test. ( H and I ) Bar plots indicate expression of Na V 1.7 and Na V 1.8 in CGRP and proprioceptor DRG subpopulations, from scRNA-seq data. p values compare cells in an unpaired t-test. ( J and K ) Hot plate and Hargreaves tests indicate that paw withdrawal latencies in response to noxious heat was significantly lengthened in Ptges3-cKO mice. n = 14, 20 mice (hot plate); 32, 17 mice (Hargreaves). Control littermates in (K) were either Ptges3 fl/fl or Ptges3 fl/+ mice. ( L ) Pain response following 1% PFA injection into the hind paw indicated the typical biphasic response. The first phase (acute pain) was unaffected, but the second phase (inflammatory pain) was reduced by ~40% in Ptges3-cKO mice. n = 9-11 mice (also see  ). ( M ) Rotarod assay (32 RPM, constant speed) indicates decreased fall latency in Ptges3-cKO mice. n = 18, 17 mice. ( N ) Precise foot placement is severely impacted in Ptges3-cKO mice recorded in the horizontal ladder test with unevenly placed rungs. n = 16 mice for each group. p values compare biological replicates (triangles-females, circles-males) in an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Journal: bioRxiv

    Article Title: Schwann cells promote sensory neuron excitability during development

    doi: 10.1101/2022.10.31.514415

    Figure Lengend Snippet: ( A ) UMAP visualization of DRG scRNA-seq data in Ptges3-Flox mice at P4. ( B ) Cell identity composition heatmaps of CGRP and proprioceptor DRG neuron subtype populations. Dark red indicates high relative cell density. ( C ) Number of CGRP and proprioceptor DRG neurons are reduced by half in Ptges3-cKO mice. ( D to G ) RNAscope shows expression of Neun, Pvalb, or Calca , and Na V 1.8 transcripts in lumbar DRG neurons at P0 to label proprioceptor or CGRP neurons, respectively. Pvalb + or Calca + neuron numbers are normalized to Neun + cells to calculate the percentage of proprioceptor or CGRP DRG neurons respectively. n = 3-5 mice, p values compare biological replicates in an unpaired t-test. ( H and I ) Bar plots indicate expression of Na V 1.7 and Na V 1.8 in CGRP and proprioceptor DRG subpopulations, from scRNA-seq data. p values compare cells in an unpaired t-test. ( J and K ) Hot plate and Hargreaves tests indicate that paw withdrawal latencies in response to noxious heat was significantly lengthened in Ptges3-cKO mice. n = 14, 20 mice (hot plate); 32, 17 mice (Hargreaves). Control littermates in (K) were either Ptges3 fl/fl or Ptges3 fl/+ mice. ( L ) Pain response following 1% PFA injection into the hind paw indicated the typical biphasic response. The first phase (acute pain) was unaffected, but the second phase (inflammatory pain) was reduced by ~40% in Ptges3-cKO mice. n = 9-11 mice (also see ). ( M ) Rotarod assay (32 RPM, constant speed) indicates decreased fall latency in Ptges3-cKO mice. n = 18, 17 mice. ( N ) Precise foot placement is severely impacted in Ptges3-cKO mice recorded in the horizontal ladder test with unevenly placed rungs. n = 16 mice for each group. p values compare biological replicates (triangles-females, circles-males) in an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

    Article Snippet: Antibodies used in this study include the following: Neurofilament (Sigma-Aldrich, N4142, 1:1000), goat anti-rabbit IgG polyclonal antibody (CF™ 405M, 20181, 1:1000), Ptges3 (knockout validated, Origene technologies, TA803433, 1:1000), Mbp (knockout validated , Abcam, ab7349, 1:100), Na v 1.8 (knockout validated, Neuromab, SKU 75-166, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.7 (knockout validated , Neuromab, SKU 75-103, 1:200 for tissues [ ]), Na v 1.1 (knockout validated , Alomone Labs, Asc-001, 1:1000), Na v 1.2 (knockout validated , Alomone Labs, Asc-002, 1:1000), Na v 1.6 (knockout validated , Alomone Labs, Asc-009, 1:1000), Na v 1.7 (knockout validated , Alomone Labs, Asc-008, 1:1000 for cultured cells, 1:200 for tissues [ ]), Na v 1.8 (knockout validated , Alomone Labs, ASC-016, 1:200 for tissues [ ]), Na v 1.5, knockout validated , Alomone Labs, ASC-005, 1:1000), Na v 1.9, Alomone Labs, AGP-030, 1:1000), and highly cross-absorbed Alexa Fluor 488-, 594-, or 647-labeled secondary antibodies (Thermo Fisher).

    Techniques: Expressing, Injection

    (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see  ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see  ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see  ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) Sample voltage clamp recordings show that sodium current was almost completely abolished by the Na V 1.8 inhibitor PF-24 (1 μM). Peak current was significantly reduced by PF-24 (F 1.72 =12.651, p<0.012, two-way RM ANOVA; n=7). Another Na V 1.8 inhibitor, A-803467, had a similar effect (see ). (B) PF-24 significantly altered spiking pattern (χ 2 =5.14, p=0.0233, McNemar test) and reduced firing rate (F 1,42 =11.946, p=0.011, two-way RM ANOVA; n=8). (C) PF-24 significantly increased rheobase (Z 15 =2.783, p=0.003, Wilcoxon rank test) and reduced spike height (T 15 =3.151, p=0.007, paired t-test) but did not affect resting membrane potential (T 15 =0.304, p=0.765, paired t-test). The Na V 1.7 inhibitor PF-71 had negligible effects at DIV0 (see ). (D) A computational model reproduced the effect of Na V 1.8 on spiking pattern (also see ). The PF-24 effect was simulated as a ~85% reduction in Na V 1.8 . *, p<0.05; **; p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques:

    (A) Inhibiting Na v 1.8 with PF-24 (1 μM) did not affect spiking pattern (χ 2 =0.00, p=1.00, McNemar test) and modestly reduced firing rate (F 1,54 =9.745, p= 0.012, two-way RM ANOVA, n=10) in DIV4-7 neurons. (B) PF-24 did not affect rheobase (Z 12 =0.420, p=0.685, Wilcoxon Rank test) but did reduce spike height (T12=2.939, p=0.012, paired-t-test). *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) Inhibiting Na v 1.8 with PF-24 (1 μM) did not affect spiking pattern (χ 2 =0.00, p=1.00, McNemar test) and modestly reduced firing rate (F 1,54 =9.745, p= 0.012, two-way RM ANOVA, n=10) in DIV4-7 neurons. (B) PF-24 did not affect rheobase (Z 12 =0.420, p=0.685, Wilcoxon Rank test) but did reduce spike height (T12=2.939, p=0.012, paired-t-test). *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques:

    (A) Sample voltage clamp recordings show that sodium current was reduced by the Na V 1.7 inhibitor PF-71 (30 nM) and by the Na V 1.1/1.3 inhibitor ICA (1 μM). Peak current was significantly reduced by PF-71 and ICA (F 2,192 =26.361, p<0.001, two-way RM ANOVA; n=9). (B) PF-71 and ICA both significantly altered spiking pattern (χ 2 =4.17, p=0.041 and χ 2 =7.11, p=0.0077, respectively, McNemar tests) and significantly reduced firing rate (F 1.54 =40.659, p<0.001, n=10 and F 1.78 =35.156, p<0.001, n=14, respectively, two-way RM ANOVAs). (C) PF-71 significantly increased rheobase (Z 18 =3.464, p<0.001, Wilcoxon rank test) and decreased spike height (T 18 =7.946, p<0.001, paired t-test). ICA did not significantly alter rheobase (Z 18 =1.248, p=0.225) but did reduce spike height (T 18 =3.243, p=0.005). Neither drug affected resting membrane potential (T 15 =1.681, p=0.113 for PF-71; T 18 =-1.132, p=0.272 for ICA, paired t-test). The Na V 1.8 antagonist PF-24 had negligible effects at DIV4-7 (see  ). (D) A computational model reproduced the combined effects of Na V 1.3 and Na V 1.7 on spiking pattern (also see  ). PF-71 effect was simulated as a 70% reduction in Na V 1.7 . ICA effect was simulated as a 90% reduction in Na V 1.3 . *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) Sample voltage clamp recordings show that sodium current was reduced by the Na V 1.7 inhibitor PF-71 (30 nM) and by the Na V 1.1/1.3 inhibitor ICA (1 μM). Peak current was significantly reduced by PF-71 and ICA (F 2,192 =26.361, p<0.001, two-way RM ANOVA; n=9). (B) PF-71 and ICA both significantly altered spiking pattern (χ 2 =4.17, p=0.041 and χ 2 =7.11, p=0.0077, respectively, McNemar tests) and significantly reduced firing rate (F 1.54 =40.659, p<0.001, n=10 and F 1.78 =35.156, p<0.001, n=14, respectively, two-way RM ANOVAs). (C) PF-71 significantly increased rheobase (Z 18 =3.464, p<0.001, Wilcoxon rank test) and decreased spike height (T 18 =7.946, p<0.001, paired t-test). ICA did not significantly alter rheobase (Z 18 =1.248, p=0.225) but did reduce spike height (T 18 =3.243, p=0.005). Neither drug affected resting membrane potential (T 15 =1.681, p=0.113 for PF-71; T 18 =-1.132, p=0.272 for ICA, paired t-test). The Na V 1.8 antagonist PF-24 had negligible effects at DIV4-7 (see ). (D) A computational model reproduced the combined effects of Na V 1.3 and Na V 1.7 on spiking pattern (also see ). PF-71 effect was simulated as a 70% reduction in Na V 1.7 . ICA effect was simulated as a 90% reduction in Na V 1.3 . *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and B.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques:

    (A) The computational model predicts that the Na V 1.8 conductance, which is “necessary” for repetitive spiking at DIV0 can, in principle, be replaced by Na V 1.7 (left), and vice versa at DIV4-7 (right). (B) Replacement experiments involved inhibiting native channels pharmacologically and then introducing virtual conductances using dynamic clamp. At DIV0 (left), inhibiting native Na V 1.8 (with PF-24) converted neurons to transient spiking, but introducing virtual Na V 1.7 reverted neurons to repetitive spiking (in 3 of 3 neurons tested). At DIV4-7, inhibiting native Na V 1.7 (with PF-71) converted the neuron to transient spiking, but introducing virtual Na V 1.8 reverted neurons to repetitive spiking (in 4 of 4 neurons tested). Parameters for virtual channels were identical to simulations except for the maximal conductance density, which was titrated in each cell. (C) Voltage (top) for first (left) and second (right) spikes in the DIV0 model aligned with voltage activation curves for each Na V isoform (bottom). Dashed line shows voltage threshold (defined as V where dV/dt reaches 5 mV/ms). (D) Conductance plotted against voltage to create a phase portrait (top) showing Na V conductance at different phases of the spike. Inset shows full voltage range; main graph zooms in on voltages near threshold. Bottom plots show current plotted over the same voltage range. For the first spike, Na V 1.7 (orange) mediates nearly all perithreshold inward current. For the second spike, voltage threshold is increased and Na V 1.8 (green) mediates nearly all perithreshold inward current because Na V 1.7 has inactivated (see  ). ( E-F ) In the DIV4-7 model, Na V 1.7 (orange) and Na V 1.3 (maroon) contribute to initiation of all spikes whereas the contribution of Na V 1.8 is negligible.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) The computational model predicts that the Na V 1.8 conductance, which is “necessary” for repetitive spiking at DIV0 can, in principle, be replaced by Na V 1.7 (left), and vice versa at DIV4-7 (right). (B) Replacement experiments involved inhibiting native channels pharmacologically and then introducing virtual conductances using dynamic clamp. At DIV0 (left), inhibiting native Na V 1.8 (with PF-24) converted neurons to transient spiking, but introducing virtual Na V 1.7 reverted neurons to repetitive spiking (in 3 of 3 neurons tested). At DIV4-7, inhibiting native Na V 1.7 (with PF-71) converted the neuron to transient spiking, but introducing virtual Na V 1.8 reverted neurons to repetitive spiking (in 4 of 4 neurons tested). Parameters for virtual channels were identical to simulations except for the maximal conductance density, which was titrated in each cell. (C) Voltage (top) for first (left) and second (right) spikes in the DIV0 model aligned with voltage activation curves for each Na V isoform (bottom). Dashed line shows voltage threshold (defined as V where dV/dt reaches 5 mV/ms). (D) Conductance plotted against voltage to create a phase portrait (top) showing Na V conductance at different phases of the spike. Inset shows full voltage range; main graph zooms in on voltages near threshold. Bottom plots show current plotted over the same voltage range. For the first spike, Na V 1.7 (orange) mediates nearly all perithreshold inward current. For the second spike, voltage threshold is increased and Na V 1.8 (green) mediates nearly all perithreshold inward current because Na V 1.7 has inactivated (see ). ( E-F ) In the DIV4-7 model, Na V 1.7 (orange) and Na V 1.3 (maroon) contribute to initiation of all spikes whereas the contribution of Na V 1.8 is negligible.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques: Activation Assay

    (A) Sample response at DIV0 showing that a virtual Na V 1.8 conductance applied with dynamic clamp restored repetitive spiking after inhibiting native Na V 1.8 channels with PF-24. This restoration was repeated in 3 of 3 neurons tested. (B) Sample recording at DIV4-7 showing that a virtual Na V 1.7 conductance restored repetitive spiking after inhibiting native Na V 1.7 channels with PF-71. This restoration was repeated in 4 of 4 neurons tested.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) Sample response at DIV0 showing that a virtual Na V 1.8 conductance applied with dynamic clamp restored repetitive spiking after inhibiting native Na V 1.8 channels with PF-24. This restoration was repeated in 3 of 3 neurons tested. (B) Sample recording at DIV4-7 showing that a virtual Na V 1.7 conductance restored repetitive spiking after inhibiting native Na V 1.7 channels with PF-71. This restoration was repeated in 4 of 4 neurons tested.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques:

    (A) In the DIV0 model, Na V 1.7 contributed to the first spike but its inactivation meant that all subsequent spikes relied exclusively on Na V 1.8. (B) In the DIV4-7 model, despite some inactivation of Na V 1.3 (red) and Na V 1.7 (green), the remaining current was still large enough to produce a net inward current sufficient to support repetitive spiking.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) In the DIV0 model, Na V 1.7 contributed to the first spike but its inactivation meant that all subsequent spikes relied exclusively on Na V 1.8. (B) In the DIV4-7 model, despite some inactivation of Na V 1.3 (red) and Na V 1.7 (green), the remaining current was still large enough to produce a net inward current sufficient to support repetitive spiking.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques:

    (A) Both Na V 1.8 and Na V 1.7 mRNA levels (relative to a housekeeping gene (HKG), see Methods) decreased significantly between DIV0 and DIV4-7 (factor 1: time, F 1,12 =56.677, p<0.001, factor 2: Na V isoform, F 1,12 =17.952, p=0.001, two-way ANOVA and Student-Newman-Keuls post-hoc tests on log transformed data, n=4 mice per time point) but more so for Na V 1.8 than for Na V 1.7 (interaction: time x isoform, F 1,12 = 11.455, p=0.005). The differential reduction translated into a significantly higher Na V 1.8: Na V 1.7 ratio on DIV0 than at DIV4-7 (T 6 =21.375, p<0.001, unpaired t-test). These changes may account for Na V 1.8 becoming unnecessary for repetitive spiking at DIV4-7 but cannot account for Na V 1.7 becoming necessary. (B) Immunoreactivity (IR) for Na V 1.8 protein exceeded Na V 1.7-IR at DIV0, but the opposite was true on DIV4-7. Na V -IR was measured relative to YFP-IR in the same cell, and then each cell’s Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the co-processed coverslip (left) or average Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the same animal (right). Both ratios were >1 at DIV0 but decreased significantly at DIV4-7 (U=78, p<0.001, n=37 for DIV0, n=40 for DIV4-7, Mann-Whitney test (left) and T 6 =4.046, p=0.007, unpaired t-test (right)). (C) Chronically applied cercosporamide (10 μM) mitigated the changes in Na V 1.8- and Na V 1.7-IR at DIV5 (Na V 1.8: H 3 =157.95, p<0.001; Na V 1.7: H 3 =80.662, p<0.001; One-way ANOVA on ranks, Dunn’s post-hoc tests, p<0.05 for all pairs). Panel on the right shows data normalized to baseline (DIV0) to emphasize relative changes.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: (A) Both Na V 1.8 and Na V 1.7 mRNA levels (relative to a housekeeping gene (HKG), see Methods) decreased significantly between DIV0 and DIV4-7 (factor 1: time, F 1,12 =56.677, p<0.001, factor 2: Na V isoform, F 1,12 =17.952, p=0.001, two-way ANOVA and Student-Newman-Keuls post-hoc tests on log transformed data, n=4 mice per time point) but more so for Na V 1.8 than for Na V 1.7 (interaction: time x isoform, F 1,12 = 11.455, p=0.005). The differential reduction translated into a significantly higher Na V 1.8: Na V 1.7 ratio on DIV0 than at DIV4-7 (T 6 =21.375, p<0.001, unpaired t-test). These changes may account for Na V 1.8 becoming unnecessary for repetitive spiking at DIV4-7 but cannot account for Na V 1.7 becoming necessary. (B) Immunoreactivity (IR) for Na V 1.8 protein exceeded Na V 1.7-IR at DIV0, but the opposite was true on DIV4-7. Na V -IR was measured relative to YFP-IR in the same cell, and then each cell’s Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the co-processed coverslip (left) or average Na V 1.8:YFP ratio was considered relative to the average Na V 1.7:YFP ratio in the same animal (right). Both ratios were >1 at DIV0 but decreased significantly at DIV4-7 (U=78, p<0.001, n=37 for DIV0, n=40 for DIV4-7, Mann-Whitney test (left) and T 6 =4.046, p=0.007, unpaired t-test (right)). (C) Chronically applied cercosporamide (10 μM) mitigated the changes in Na V 1.8- and Na V 1.7-IR at DIV5 (Na V 1.8: H 3 =157.95, p<0.001; Na V 1.7: H 3 =80.662, p<0.001; One-way ANOVA on ranks, Dunn’s post-hoc tests, p<0.05 for all pairs). Panel on the right shows data normalized to baseline (DIV0) to emphasize relative changes.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques: Transformation Assay, MANN-WHITNEY

    ( A ) Sample responses in DIV0 neurons from mice injected with CFA three days earlier. In 12 cells tested, PF-71 converted 5 neurons to transient spiking ( i ), encouraged repetitive spiking in 4 neurons ( ii ), and had no effect in 3 neurons ( iii ), thus highlighting increased heterogeneity after CFA. ( B ) At DIV0, the effect of PF-71 differed significantly between CFA and control neurons, converting 42% (5 of 12) CFA neurons from repetitive to transient spiking vs 0% (0 of 9) control neurons (p=0.0451, Fisher Exact test). Applying PF-24 to neurons that continued to spike repetitively after PF-71 had little effect on CFA neuron, converting only 13% (1 of 7) of CFA neurons vs 88% (7 of 8) of control neurons (p=0.001, Fisher Exact test). Together these results argue that Na V 1.7 contributes more and Na V 1.8 contributes less to nociceptor excitability after inflammation. (C) At DIV0, PF-71 significantly increased resting membrane potential (T 11 =-3.530, p=0.005, paired t-test) and rheobase (Z 11 =2.186, p=0.024, Wilcoxon rank test), and significantly decreased spike height (T 11 =4.413, p=0.001, paired t-test) in CFA neurons. Further addition of PF-24 significantly changed rheobase (Z 9 =2.176, p=0.023, Wilcoxon rank test) and action potential amplitude (T 9 =3.237, p=0.01, paired t-test) but did not affect resting membrane potential (T 9 =1.049, p=0.321, paired t-test). (D) Paw inflammation caused by CFA significantly altered thermal sensitivity (Hargreaves: F 5,65 =19.556, p<0.001, two-way RM ANOVA) and mechanical sensitivity (von Frey: F 4,52 =16.786, p<0.001). When given three days after CFA, PF-71 significantly reversed the altered sensitivities (Hargreaves: T 8 =-7.296, p<0.001; von Frey: T 8 =-4.341, p=0.002; paired t-tests) but had no effect in naive mice (Hargreaves: T 5 =-0.141, p=0.894; von Frey: T 5 =1.000, p=0.363). Insets show values for each animal before and 2 hours after PF-71 injection. *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests.

    Journal: bioRxiv

    Article Title: Equivalent excitability through different sodium channel subtypes and implications for analgesia by subtype-selective drugs

    doi: 10.1101/2022.10.04.510784

    Figure Lengend Snippet: ( A ) Sample responses in DIV0 neurons from mice injected with CFA three days earlier. In 12 cells tested, PF-71 converted 5 neurons to transient spiking ( i ), encouraged repetitive spiking in 4 neurons ( ii ), and had no effect in 3 neurons ( iii ), thus highlighting increased heterogeneity after CFA. ( B ) At DIV0, the effect of PF-71 differed significantly between CFA and control neurons, converting 42% (5 of 12) CFA neurons from repetitive to transient spiking vs 0% (0 of 9) control neurons (p=0.0451, Fisher Exact test). Applying PF-24 to neurons that continued to spike repetitively after PF-71 had little effect on CFA neuron, converting only 13% (1 of 7) of CFA neurons vs 88% (7 of 8) of control neurons (p=0.001, Fisher Exact test). Together these results argue that Na V 1.7 contributes more and Na V 1.8 contributes less to nociceptor excitability after inflammation. (C) At DIV0, PF-71 significantly increased resting membrane potential (T 11 =-3.530, p=0.005, paired t-test) and rheobase (Z 11 =2.186, p=0.024, Wilcoxon rank test), and significantly decreased spike height (T 11 =4.413, p=0.001, paired t-test) in CFA neurons. Further addition of PF-24 significantly changed rheobase (Z 9 =2.176, p=0.023, Wilcoxon rank test) and action potential amplitude (T 9 =3.237, p=0.01, paired t-test) but did not affect resting membrane potential (T 9 =1.049, p=0.321, paired t-test). (D) Paw inflammation caused by CFA significantly altered thermal sensitivity (Hargreaves: F 5,65 =19.556, p<0.001, two-way RM ANOVA) and mechanical sensitivity (von Frey: F 4,52 =16.786, p<0.001). When given three days after CFA, PF-71 significantly reversed the altered sensitivities (Hargreaves: T 8 =-7.296, p<0.001; von Frey: T 8 =-4.341, p=0.002; paired t-tests) but had no effect in naive mice (Hargreaves: T 5 =-0.141, p=0.894; von Frey: T 5 =1.000, p=0.363). Insets show values for each animal before and 2 hours after PF-71 injection. *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests.

    Article Snippet: After another 3x rinse with PBS, neurons were treated with 10% normal goat serum for 30 min followed with rabbit primary Na V 1.7 antibody (1:200, ASC-008, Alomone) or Na V 1.8 antibody (1:200, ASC-028, Alomone) in PBS with 0.1% Tritween-20 and 1% BSA for 1 h. For some of the coverslips, primary antibodies were replaced with control peptides (ASC008AG1040 for Na V 1.7 and ASC016AG0640 for Na V 1.8) provided by Alomone as negative controls.

    Techniques: Injection