anti nav1 7 scn9a atto fluor 633 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti nav1 7 scn9a atto fluor 633 antibody
    Enhanced heat pain sensitivity in <t>Scn9a</t> R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.
    Anti Nav1 7 Scn9a Atto Fluor 633 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
    https://www.bioz.com/result/anti nav1 7 scn9a atto fluor 633 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nav1 7 scn9a atto fluor 633 antibody - by Bioz Stars, 2022-10
    94/100 stars

    Images

    1) Product Images from "The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice"

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2022.913990

    Enhanced heat pain sensitivity in Scn9a R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.
    Figure Legend Snippet: Enhanced heat pain sensitivity in Scn9a R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.

    Techniques Used: Mouse Assay, Tail Flick Test, Mutagenesis

    Nav1.7 protein expression of DRG and sciatic nerve in Scn9a R 185 H mutant mice. (A) Representative images of PGP9.5 (green) and Nav1.7 (red) fluorescent immunostaining for all neurons (PGP9.5-positive) and Nav1.7-positive neurons in lumbar DRG of wt animals. Scale bar 50 μm. (B) Size distribution for Nav1.7-positive (red) and negative (white) neuron cross-sectional areas in wt , het, and homo female and male mice. Females: n = 5/group; male: n = 3/group. Results are shown as means ± SEM. The two-way ANOVAs for genotype and neuron size effects in each sex showed a size effect and no genotype effect. (C) Representative images of fluorescent PGP9.5 (green) and Nav1.7 (red) immunostaining of sciatic nerves of wt animals. Scale bar of 25 μm. (D) Mean fluorescence density Nav1.7 immunostaining of sciatic nerves in wt, het, and homo female and male mice. Females, n = 4–5/group; males, n = 3/group. Results are shown as means ± SEM. The two-way ANOVA for genotype and sex indicated a sex effect and no genotype effect. The P value of 0.009 is for sex effect. See Supplementary Table 6 for statistics.
    Figure Legend Snippet: Nav1.7 protein expression of DRG and sciatic nerve in Scn9a R 185 H mutant mice. (A) Representative images of PGP9.5 (green) and Nav1.7 (red) fluorescent immunostaining for all neurons (PGP9.5-positive) and Nav1.7-positive neurons in lumbar DRG of wt animals. Scale bar 50 μm. (B) Size distribution for Nav1.7-positive (red) and negative (white) neuron cross-sectional areas in wt , het, and homo female and male mice. Females: n = 5/group; male: n = 3/group. Results are shown as means ± SEM. The two-way ANOVAs for genotype and neuron size effects in each sex showed a size effect and no genotype effect. (C) Representative images of fluorescent PGP9.5 (green) and Nav1.7 (red) immunostaining of sciatic nerves of wt animals. Scale bar of 25 μm. (D) Mean fluorescence density Nav1.7 immunostaining of sciatic nerves in wt, het, and homo female and male mice. Females, n = 4–5/group; males, n = 3/group. Results are shown as means ± SEM. The two-way ANOVA for genotype and sex indicated a sex effect and no genotype effect. The P value of 0.009 is for sex effect. See Supplementary Table 6 for statistics.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Immunostaining, Fluorescence

    Scn9a R 185 H mice show enhanced sensitivity to cooling and touch stimuli. (A,B). Homozygous mutant males show increased duration and number of paw reactions in the acetone test. (C). Male mutant mice show a trend to reduced number of paw lifts on the cold plate. (D). Mutant mice display lower sensitivity threshold in the von Frey test. (E). Mutant mice show no difference to wt mice in the tail pressure test. Results are shown as means ± SEM. Acetone duration of paw reactions and number of paw reactions: females: wt, n = 15; Het, n = 13; Homo, n = 13; males: wt, n = 14; Het, n = 13; Homo, n = 12. Cold plate: females: wt, n = 15; Het, n = 15; Homo, n = 13; males: wt, n = 15; Het, n = 14; Homo, n = 12. Von Frey: females: wt, n = 13; Het, n = 13; Homo, n = 10; males: wt, n = 14; Het, n = 14; Homo, n = 11. Tail pressure: females: wt, n = 13; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 10 , 11 for statistics.
    Figure Legend Snippet: Scn9a R 185 H mice show enhanced sensitivity to cooling and touch stimuli. (A,B). Homozygous mutant males show increased duration and number of paw reactions in the acetone test. (C). Male mutant mice show a trend to reduced number of paw lifts on the cold plate. (D). Mutant mice display lower sensitivity threshold in the von Frey test. (E). Mutant mice show no difference to wt mice in the tail pressure test. Results are shown as means ± SEM. Acetone duration of paw reactions and number of paw reactions: females: wt, n = 15; Het, n = 13; Homo, n = 13; males: wt, n = 14; Het, n = 13; Homo, n = 12. Cold plate: females: wt, n = 15; Het, n = 15; Homo, n = 13; males: wt, n = 15; Het, n = 14; Homo, n = 12. Von Frey: females: wt, n = 13; Het, n = 13; Homo, n = 10; males: wt, n = 14; Het, n = 14; Homo, n = 11. Tail pressure: females: wt, n = 13; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 10 , 11 for statistics.

    Techniques Used: Mouse Assay, Mutagenesis

    Scn9a R 185 H mutant mice show spontaneous ongoing pain. Ongoing pain was assessed in a conditioned place preference test. After pre-test/habituation for chamber preference on days 1 to 3, conditioning was made on day 4 by intrathecal treatment with either saline solution in the morning or clonidine solution in the afternoon with a restriction in one chamber. On day 5, the animals were free to explore the chambers and the time spent in each chamber was recorded for 30 min. (A) Females. (B) Males. The time spent in each chamber before conditioning (pre-test) and after conditioning with clonidine or saline (test) are shown. Homozygous mice of both sexes spent more time in clonidine-paired chamber while wt mice spent on average equal time in saline-paired and clonidine-paired chambers. Dots represent individual data points. females: wt, n = 18; Homo, n = 18; males: wt, n = 10; Homo, n = 10. Two-way ANOVA for treatment and trial (pretest and test) on each genotype and sex group, Sidak’s multiple comparison test. P = values for comparing pre-test and test values are shown. Refer to Supplementary Table 12 for statistics.
    Figure Legend Snippet: Scn9a R 185 H mutant mice show spontaneous ongoing pain. Ongoing pain was assessed in a conditioned place preference test. After pre-test/habituation for chamber preference on days 1 to 3, conditioning was made on day 4 by intrathecal treatment with either saline solution in the morning or clonidine solution in the afternoon with a restriction in one chamber. On day 5, the animals were free to explore the chambers and the time spent in each chamber was recorded for 30 min. (A) Females. (B) Males. The time spent in each chamber before conditioning (pre-test) and after conditioning with clonidine or saline (test) are shown. Homozygous mice of both sexes spent more time in clonidine-paired chamber while wt mice spent on average equal time in saline-paired and clonidine-paired chambers. Dots represent individual data points. females: wt, n = 18; Homo, n = 18; males: wt, n = 10; Homo, n = 10. Two-way ANOVA for treatment and trial (pretest and test) on each genotype and sex group, Sidak’s multiple comparison test. P = values for comparing pre-test and test values are shown. Refer to Supplementary Table 12 for statistics.

    Techniques Used: Mutagenesis, Mouse Assay, Conditioned Place Preference

    Generation of Scn9a R 185 H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for Bsp HI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a R 185 H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t -test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to Supplementary Table 5 for statistics.
    Figure Legend Snippet: Generation of Scn9a R 185 H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for Bsp HI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a R 185 H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t -test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to Supplementary Table 5 for statistics.

    Techniques Used: Mutagenesis, Mouse Assay, Expressing, Genotyping Assay, Sequencing, Two Tailed Test

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    Alomone Labs anti nav1 7 scn9a atto fluor 633 antibody
    Enhanced heat pain sensitivity in <t>Scn9a</t> R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.
    Anti Nav1 7 Scn9a Atto Fluor 633 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
    https://www.bioz.com/result/anti nav1 7 scn9a atto fluor 633 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nav1 7 scn9a atto fluor 633 antibody - by Bioz Stars, 2022-10
    94/100 stars
      Buy from Supplier

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    Enhanced heat pain sensitivity in Scn9a R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    doi: 10.3389/fnmol.2022.913990

    Figure Lengend Snippet: Enhanced heat pain sensitivity in Scn9a R 185 H mice. (A) Wild-type and Scn9a R 185 H mice showed comparable first response latency on the hot plate. (B) Homozygous Scn9a R 185 H females had decreased jump latency in 56°C Hot Plate. (C) Homozygous Scn9a R 185 H female mice showed more coping reactions on the 52°C hot plate. (D) Heterozygous and homozygous Scn9a R 185 H females had lower tail latency in the tail flick test. (E) No difference was found between wt and mutant mice in the Hargreaves test. (F) Control wt and mutant mice showed the same temperature preference. Results are shown as means ± SEM. Hot plate, females: wt, n = 16; Het, n = 16; Homo, n = 15; males: wt, n = 13; Het, n = 14; Homo, n = 11. Tail flick: females: wt, n = 15; Het, n = 15; Homo, n = 14; males: wt, n = 14; Het, n = 14; Homo, n = 11. Hargreaves: females: wt, n = 14; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. Temperature preference: females: wt, n = 12; Homo, n = 12; males: wt, n = 11; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests for hot plate, tail flick, and Hargreaves tests; two-way ANOVA for the thermal preference ring test. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 7 – 9 for statistics.

    Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

    Techniques: Mouse Assay, Tail Flick Test, Mutagenesis

    Nav1.7 protein expression of DRG and sciatic nerve in Scn9a R 185 H mutant mice. (A) Representative images of PGP9.5 (green) and Nav1.7 (red) fluorescent immunostaining for all neurons (PGP9.5-positive) and Nav1.7-positive neurons in lumbar DRG of wt animals. Scale bar 50 μm. (B) Size distribution for Nav1.7-positive (red) and negative (white) neuron cross-sectional areas in wt , het, and homo female and male mice. Females: n = 5/group; male: n = 3/group. Results are shown as means ± SEM. The two-way ANOVAs for genotype and neuron size effects in each sex showed a size effect and no genotype effect. (C) Representative images of fluorescent PGP9.5 (green) and Nav1.7 (red) immunostaining of sciatic nerves of wt animals. Scale bar of 25 μm. (D) Mean fluorescence density Nav1.7 immunostaining of sciatic nerves in wt, het, and homo female and male mice. Females, n = 4–5/group; males, n = 3/group. Results are shown as means ± SEM. The two-way ANOVA for genotype and sex indicated a sex effect and no genotype effect. The P value of 0.009 is for sex effect. See Supplementary Table 6 for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    doi: 10.3389/fnmol.2022.913990

    Figure Lengend Snippet: Nav1.7 protein expression of DRG and sciatic nerve in Scn9a R 185 H mutant mice. (A) Representative images of PGP9.5 (green) and Nav1.7 (red) fluorescent immunostaining for all neurons (PGP9.5-positive) and Nav1.7-positive neurons in lumbar DRG of wt animals. Scale bar 50 μm. (B) Size distribution for Nav1.7-positive (red) and negative (white) neuron cross-sectional areas in wt , het, and homo female and male mice. Females: n = 5/group; male: n = 3/group. Results are shown as means ± SEM. The two-way ANOVAs for genotype and neuron size effects in each sex showed a size effect and no genotype effect. (C) Representative images of fluorescent PGP9.5 (green) and Nav1.7 (red) immunostaining of sciatic nerves of wt animals. Scale bar of 25 μm. (D) Mean fluorescence density Nav1.7 immunostaining of sciatic nerves in wt, het, and homo female and male mice. Females, n = 4–5/group; males, n = 3/group. Results are shown as means ± SEM. The two-way ANOVA for genotype and sex indicated a sex effect and no genotype effect. The P value of 0.009 is for sex effect. See Supplementary Table 6 for statistics.

    Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

    Techniques: Expressing, Mutagenesis, Mouse Assay, Immunostaining, Fluorescence

    Scn9a R 185 H mice show enhanced sensitivity to cooling and touch stimuli. (A,B). Homozygous mutant males show increased duration and number of paw reactions in the acetone test. (C). Male mutant mice show a trend to reduced number of paw lifts on the cold plate. (D). Mutant mice display lower sensitivity threshold in the von Frey test. (E). Mutant mice show no difference to wt mice in the tail pressure test. Results are shown as means ± SEM. Acetone duration of paw reactions and number of paw reactions: females: wt, n = 15; Het, n = 13; Homo, n = 13; males: wt, n = 14; Het, n = 13; Homo, n = 12. Cold plate: females: wt, n = 15; Het, n = 15; Homo, n = 13; males: wt, n = 15; Het, n = 14; Homo, n = 12. Von Frey: females: wt, n = 13; Het, n = 13; Homo, n = 10; males: wt, n = 14; Het, n = 14; Homo, n = 11. Tail pressure: females: wt, n = 13; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 10 , 11 for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    doi: 10.3389/fnmol.2022.913990

    Figure Lengend Snippet: Scn9a R 185 H mice show enhanced sensitivity to cooling and touch stimuli. (A,B). Homozygous mutant males show increased duration and number of paw reactions in the acetone test. (C). Male mutant mice show a trend to reduced number of paw lifts on the cold plate. (D). Mutant mice display lower sensitivity threshold in the von Frey test. (E). Mutant mice show no difference to wt mice in the tail pressure test. Results are shown as means ± SEM. Acetone duration of paw reactions and number of paw reactions: females: wt, n = 15; Het, n = 13; Homo, n = 13; males: wt, n = 14; Het, n = 13; Homo, n = 12. Cold plate: females: wt, n = 15; Het, n = 15; Homo, n = 13; males: wt, n = 15; Het, n = 14; Homo, n = 12. Von Frey: females: wt, n = 13; Het, n = 13; Homo, n = 10; males: wt, n = 14; Het, n = 14; Homo, n = 11. Tail pressure: females: wt, n = 13; Het, n = 13; Homo, n = 11; males: wt, n = 14; Het, n = 14; Homo, n = 11. One-way ANOVA on females and males and Dunnett’s multiple comparison tests. P -values for genotype difference are shown when significant or close to significance. See Supplementary Tables 10 , 11 for statistics.

    Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

    Techniques: Mouse Assay, Mutagenesis

    Scn9a R 185 H mutant mice show spontaneous ongoing pain. Ongoing pain was assessed in a conditioned place preference test. After pre-test/habituation for chamber preference on days 1 to 3, conditioning was made on day 4 by intrathecal treatment with either saline solution in the morning or clonidine solution in the afternoon with a restriction in one chamber. On day 5, the animals were free to explore the chambers and the time spent in each chamber was recorded for 30 min. (A) Females. (B) Males. The time spent in each chamber before conditioning (pre-test) and after conditioning with clonidine or saline (test) are shown. Homozygous mice of both sexes spent more time in clonidine-paired chamber while wt mice spent on average equal time in saline-paired and clonidine-paired chambers. Dots represent individual data points. females: wt, n = 18; Homo, n = 18; males: wt, n = 10; Homo, n = 10. Two-way ANOVA for treatment and trial (pretest and test) on each genotype and sex group, Sidak’s multiple comparison test. P = values for comparing pre-test and test values are shown. Refer to Supplementary Table 12 for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    doi: 10.3389/fnmol.2022.913990

    Figure Lengend Snippet: Scn9a R 185 H mutant mice show spontaneous ongoing pain. Ongoing pain was assessed in a conditioned place preference test. After pre-test/habituation for chamber preference on days 1 to 3, conditioning was made on day 4 by intrathecal treatment with either saline solution in the morning or clonidine solution in the afternoon with a restriction in one chamber. On day 5, the animals were free to explore the chambers and the time spent in each chamber was recorded for 30 min. (A) Females. (B) Males. The time spent in each chamber before conditioning (pre-test) and after conditioning with clonidine or saline (test) are shown. Homozygous mice of both sexes spent more time in clonidine-paired chamber while wt mice spent on average equal time in saline-paired and clonidine-paired chambers. Dots represent individual data points. females: wt, n = 18; Homo, n = 18; males: wt, n = 10; Homo, n = 10. Two-way ANOVA for treatment and trial (pretest and test) on each genotype and sex group, Sidak’s multiple comparison test. P = values for comparing pre-test and test values are shown. Refer to Supplementary Table 12 for statistics.

    Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

    Techniques: Mutagenesis, Mouse Assay, Conditioned Place Preference

    Generation of Scn9a R 185 H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for Bsp HI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a R 185 H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t -test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to Supplementary Table 5 for statistics.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice

    doi: 10.3389/fnmol.2022.913990

    Figure Lengend Snippet: Generation of Scn9a R 185 H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for Bsp HI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a R 185 H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t -test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to Supplementary Table 5 for statistics.

    Article Snippet: The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam).

    Techniques: Mutagenesis, Mouse Assay, Expressing, Genotyping Assay, Sequencing, Two Tailed Test