α latrotoxin  (Alomone Labs)


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    Structured Review

    Alomone Labs α latrotoxin
    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM <t>α-latrotoxin</t> (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.
    α Latrotoxin, 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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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α latrotoxin - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Loss of transforming growth factor-beta 2 leads to impairment of central synapse function"

    Article Title: Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

    Journal: Neural Development

    doi: 10.1186/1749-8104-3-25

    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.
    Figure Legend Snippet: Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.

    Techniques Used: Transmission Assay, Knock-Out

    α latrotoxin  (Alomone Labs)


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    Structured Review

    Alomone Labs α latrotoxin
    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM <t>α-latrotoxin</t> (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.
    α Latrotoxin, 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/α latrotoxin/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α latrotoxin - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Loss of transforming growth factor-beta 2 leads to impairment of central synapse function"

    Article Title: Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

    Journal: Neural Development

    doi: 10.1186/1749-8104-3-25

    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.
    Figure Legend Snippet: Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.

    Techniques Used: Transmission Assay, Knock-Out

    α ltx  (Alomone Labs)


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    Structured Review

    Alomone Labs α ltx
    Time course of NMJ degeneration and regeneration by <t>α-LTx</t> reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.
    α Ltx, 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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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α ltx - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration"

    Article Title: Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23031186

    Time course of NMJ degeneration and regeneration by α-LTx reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.
    Figure Legend Snippet: Time course of NMJ degeneration and regeneration by α-LTx reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.

    Techniques Used: Immunostaining, Activity Assay, Injection, Staining, Isolation, Amplification, Digital PCR

    The CRHR2 receptor participates in the structural and functional recovery of the NMJ. ( a ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or the CRHR2 antagonist A2B up to 96 h after α-LTx injection, and the analysis performed to assess the functional and structural status of the NMJ. ( b ) Electrophysiological recordings of evoked EPPs from intoxicated soleus muscles after either vehicle of A2B administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. ** p < 0.01, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( c ) Representative immunostaining and ( d ) quantification performed on soleus muscles after intoxication with α-LTx and treatment with either vehicle or A2B. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (lack or little VAMP-1 signal). The right panel shows a magnification. Scale bars: 50 µm. Bars in ( d ) represent the mean± SD percentage of regenerated NMJs. ** p < 0.01 by Student’s t -test.
    Figure Legend Snippet: The CRHR2 receptor participates in the structural and functional recovery of the NMJ. ( a ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or the CRHR2 antagonist A2B up to 96 h after α-LTx injection, and the analysis performed to assess the functional and structural status of the NMJ. ( b ) Electrophysiological recordings of evoked EPPs from intoxicated soleus muscles after either vehicle of A2B administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. ** p < 0.01, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( c ) Representative immunostaining and ( d ) quantification performed on soleus muscles after intoxication with α-LTx and treatment with either vehicle or A2B. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (lack or little VAMP-1 signal). The right panel shows a magnification. Scale bars: 50 µm. Bars in ( d ) represent the mean± SD percentage of regenerated NMJs. ** p < 0.01 by Student’s t -test.

    Techniques Used: Functional Assay, Injection, Immunostaining

    The UCN2-CRHR2 axis promotes axonal growth of cultured SCMNs and NMJ regeneration after acute damage. ( a ) Representative images of neurofilament staining used as reporter for axon length in SCMNs cultured for 24 h either in normal culture medium (NC) or NC supplemented with UCN2, A2B or their combination. ( b ) Quantification of the average axonal length after the indicated treatments. Three independent experiments were performed, and at least 100 neurons per condition measured. Bars represent mean ± SD. Statical analysis was performed with ANOVA followed by post hoc Tukey test; *** p < 0.001, ** p < 0.01, ns p > 0.05. ( c ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or recombinant UCN2 for 72 h after α-LTx treatment, followed by functional and structural assessment of NMJ status. ( d ) Electrophysiological assessment of NMJ function by evoked EPPs recordings following either vehicle or UCN2 administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. * p < 0.05, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( e ) Representative immunostaining and ( f ) quantification performed on soleus muscles after intoxication and treatment with either vehicle or UCN2. Regenerated NMJs are positive for both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (absent or faint VAMP1 signal). Right panels are magnifications of the indicated NMJs. Scale bars: 50 µm, 20 µm for magnification. Each bar in ( f ) represents the mean ± SD percentage of recovered NMJs. ** p < 0.01 by Student’s t -test.
    Figure Legend Snippet: The UCN2-CRHR2 axis promotes axonal growth of cultured SCMNs and NMJ regeneration after acute damage. ( a ) Representative images of neurofilament staining used as reporter for axon length in SCMNs cultured for 24 h either in normal culture medium (NC) or NC supplemented with UCN2, A2B or their combination. ( b ) Quantification of the average axonal length after the indicated treatments. Three independent experiments were performed, and at least 100 neurons per condition measured. Bars represent mean ± SD. Statical analysis was performed with ANOVA followed by post hoc Tukey test; *** p < 0.001, ** p < 0.01, ns p > 0.05. ( c ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or recombinant UCN2 for 72 h after α-LTx treatment, followed by functional and structural assessment of NMJ status. ( d ) Electrophysiological assessment of NMJ function by evoked EPPs recordings following either vehicle or UCN2 administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. * p < 0.05, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( e ) Representative immunostaining and ( f ) quantification performed on soleus muscles after intoxication and treatment with either vehicle or UCN2. Regenerated NMJs are positive for both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (absent or faint VAMP1 signal). Right panels are magnifications of the indicated NMJs. Scale bars: 50 µm, 20 µm for magnification. Each bar in ( f ) represents the mean ± SD percentage of recovered NMJs. ** p < 0.01 by Student’s t -test.

    Techniques Used: Cell Culture, Staining, Recombinant, Functional Assay, Immunostaining

    α ltx  (Alomone Labs)


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    Alomone Labs α ltx
    Output and assessment of phage display selections against <t>α-LTX.</t> (A) Accumulation of scFv-displaying phages from the IONTAS phage display library λ with affinity to α-LTX over three rounds of selection. An increase in CFU/ml of 375-fold and 22-fold respectively were observed between the selection rounds. (B) CFU/ml was determined for output phages from selection round two and three for binding to either α-LTX, streptavidin, or milk proteins. An increase of 30-fold between the second and third selection round was observed in CFU/ml for the phages that bound streptavidin-captured α-LTX. Only few phages with affinity to streptavidin or milk proteins were accumulated compared to phages with affinity to α-LTX.
    α Ltx, 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/α ltx/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α ltx - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Discovery of a Recombinant Human Monoclonal Immunoglobulin G Antibody Against α-Latrotoxin From the Mediterranean Black Widow Spider ( Latrodectus tredecimguttatus )"

    Article Title: Discovery of a Recombinant Human Monoclonal Immunoglobulin G Antibody Against α-Latrotoxin From the Mediterranean Black Widow Spider ( Latrodectus tredecimguttatus )

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.587825

    Output and assessment of phage display selections against α-LTX. (A) Accumulation of scFv-displaying phages from the IONTAS phage display library λ with affinity to α-LTX over three rounds of selection. An increase in CFU/ml of 375-fold and 22-fold respectively were observed between the selection rounds. (B) CFU/ml was determined for output phages from selection round two and three for binding to either α-LTX, streptavidin, or milk proteins. An increase of 30-fold between the second and third selection round was observed in CFU/ml for the phages that bound streptavidin-captured α-LTX. Only few phages with affinity to streptavidin or milk proteins were accumulated compared to phages with affinity to α-LTX.
    Figure Legend Snippet: Output and assessment of phage display selections against α-LTX. (A) Accumulation of scFv-displaying phages from the IONTAS phage display library λ with affinity to α-LTX over three rounds of selection. An increase in CFU/ml of 375-fold and 22-fold respectively were observed between the selection rounds. (B) CFU/ml was determined for output phages from selection round two and three for binding to either α-LTX, streptavidin, or milk proteins. An increase of 30-fold between the second and third selection round was observed in CFU/ml for the phages that bound streptavidin-captured α-LTX. Only few phages with affinity to streptavidin or milk proteins were accumulated compared to phages with affinity to α-LTX.

    Techniques Used: Selection, Binding Assay

    Monoclonal scFv ELISA signals against α-LTX. In total, 534 scFv clones were expressed in solution and screened for their ability to bind directly coated α-LTX. scFvs displaying a binding signal above the cut-off absorbance value of 0.5 (dotted line) at 492 nm were considered hits.
    Figure Legend Snippet: Monoclonal scFv ELISA signals against α-LTX. In total, 534 scFv clones were expressed in solution and screened for their ability to bind directly coated α-LTX. scFvs displaying a binding signal above the cut-off absorbance value of 0.5 (dotted line) at 492 nm were considered hits.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Clone Assay, Binding Assay

    Monoclonal IgG ELISA signals against (A) α-LTX and (B) L. tredecimguttatus whole venom. The binding capability of TPL0020_02_G9 to α-LTX was assessed using different concentrations of IgG. The binding specificity was evaluated by testing the binding of the IgG to three controls (binding to milk proteins, streptavidin, and neutravidin) using the highest IgG concentration (2,000 ng/ml). Each column represents the average of triplicate measurements with error bars indicating the standard deviation.
    Figure Legend Snippet: Monoclonal IgG ELISA signals against (A) α-LTX and (B) L. tredecimguttatus whole venom. The binding capability of TPL0020_02_G9 to α-LTX was assessed using different concentrations of IgG. The binding specificity was evaluated by testing the binding of the IgG to three controls (binding to milk proteins, streptavidin, and neutravidin) using the highest IgG concentration (2,000 ng/ml). Each column represents the average of triplicate measurements with error bars indicating the standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Standard Deviation

    Effect of α-LTX and its mixture with IgG TPL0020_02_G9 at 1:10 ratio (w/w) on spontaneous EPSC frequency in pyramidal neurons from mPFC. (A) Representative recording of α-LTX action. Time course of the experiment and expanded parts in control (1), after 10 min of α-LTX application (2), and after 10 μM DNQX and 100 μM D-APV application (3) are shown. α-LTX causes a strong increase of spontaneous EPSC frequency, and selective antagonists of ionotropic glutamate receptors abolish this effect. (B) Representative recording of α-LTX action in the presence of IgG TPL0020_02_G9. Strong frequency increase is seen only after 20 min of application. (C) Time development of α-LTX effect and its modulation by IgG. Spontaneous EPSC frequencies are normalized to the average control values. The development of effect is strongly delayed in the presence of IgG (red traces) compared to control with α-LTX alone (black traces). (D) Characteristic times when the 3 × SD threshold is reached or spontaneous EPSC frequency is increased four-fold for α-LTX alone (white bars) and in the presence of the IgG (red bars). The differences are significant (p < 0.05, unpaired t-test).
    Figure Legend Snippet: Effect of α-LTX and its mixture with IgG TPL0020_02_G9 at 1:10 ratio (w/w) on spontaneous EPSC frequency in pyramidal neurons from mPFC. (A) Representative recording of α-LTX action. Time course of the experiment and expanded parts in control (1), after 10 min of α-LTX application (2), and after 10 μM DNQX and 100 μM D-APV application (3) are shown. α-LTX causes a strong increase of spontaneous EPSC frequency, and selective antagonists of ionotropic glutamate receptors abolish this effect. (B) Representative recording of α-LTX action in the presence of IgG TPL0020_02_G9. Strong frequency increase is seen only after 20 min of application. (C) Time development of α-LTX effect and its modulation by IgG. Spontaneous EPSC frequencies are normalized to the average control values. The development of effect is strongly delayed in the presence of IgG (red traces) compared to control with α-LTX alone (black traces). (D) Characteristic times when the 3 × SD threshold is reached or spontaneous EPSC frequency is increased four-fold for α-LTX alone (white bars) and in the presence of the IgG (red bars). The differences are significant (p < 0.05, unpaired t-test).

    Techniques Used:

    α ltx  (Alomone Labs)


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    Alomone Labs α ltx
    Melatonin promotes nerve terminal regeneration. A, Evoked junctional potentials (EJPs) amplitude of soleus muscles 72 h after <t>α‐LTx</t> injection in the hind limb, w/o melatonin i.p. injections. Each bar represents mean ± SEM from N = 4, number of analyzed fibers: 10, * P < .05. B, Representative immunostaining and (C) quantitation performed on the same muscles in A. * P < .05. MAT is identified by syntaxin immunostaining ( green ), postsynaptic AChRs by fluorescent α‐BTx ( red ). White asterisks indicate still degenerated NMJs. Lower panels show NMJs at higher magnification. Scale bars: 20 µm upper panels, 10 µm lower panels
    α Ltx, 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 "Melatonin promotes regeneration of injured motor axons via MT 1 receptors"

    Article Title: Melatonin promotes regeneration of injured motor axons via MT 1 receptors

    Journal: Journal of Pineal Research

    doi: 10.1111/jpi.12695

    Melatonin promotes nerve terminal regeneration. A, Evoked junctional potentials (EJPs) amplitude of soleus muscles 72 h after α‐LTx injection in the hind limb, w/o melatonin i.p. injections. Each bar represents mean ± SEM from N = 4, number of analyzed fibers: 10, * P < .05. B, Representative immunostaining and (C) quantitation performed on the same muscles in A. * P < .05. MAT is identified by syntaxin immunostaining ( green ), postsynaptic AChRs by fluorescent α‐BTx ( red ). White asterisks indicate still degenerated NMJs. Lower panels show NMJs at higher magnification. Scale bars: 20 µm upper panels, 10 µm lower panels
    Figure Legend Snippet: Melatonin promotes nerve terminal regeneration. A, Evoked junctional potentials (EJPs) amplitude of soleus muscles 72 h after α‐LTx injection in the hind limb, w/o melatonin i.p. injections. Each bar represents mean ± SEM from N = 4, number of analyzed fibers: 10, * P < .05. B, Representative immunostaining and (C) quantitation performed on the same muscles in A. * P < .05. MAT is identified by syntaxin immunostaining ( green ), postsynaptic AChRs by fluorescent α‐BTx ( red ). White asterisks indicate still degenerated NMJs. Lower panels show NMJs at higher magnification. Scale bars: 20 µm upper panels, 10 µm lower panels

    Techniques Used: Injection, Immunostaining, Quantitation Assay

    MT 1 receptors are expressed at the NMJ and along the sciatic nerve and mediate the pro‐regenerative action of melatonin. A, EJPs amplitude of soleus muscles: (a) in control conditions (± luzindole); (b) +α‐LTx ± i.p. daily treatment with melatonin ± luzindole daily local injections (time point: 72 h after injury); (c) +α‐LTx ± luzindole daily local injections (time point: 96 h after injury). Each bar represents mean ± SEM from N = 4 (number of analyzed fibers: 10). * P < .05, ** P < .01, ns = not significant. F ‐value (DFn, DFd) = 13.65 (4,19); P ‐value < .0001. B, EJPs amplitude of soleus muscles: (a) in control conditions; (b) + α‐LTx ± i.p. daily melatonin; (c) +α‐LTx + i.p. daily melatonin +4P‐PDOT daily local injections; (d) +α‐LTx +ramelteon or tasimelteon daily local injections. For all conditions: time point = 72 h after injury. Each bar represents mean ± SEM from N = 4 (number of analyzed fibers: 10). * P < .05, ** P < .01, *** P < .001, ns = not significant. F ‐value (DFn, DFd) = 9.459 (4,15); P ‐value = .0005. C, MT 1 staining (mAb‐A06, red ) at LAL NMJs in controls (upper panels) and 24 h after α‐LTx injection (lower panels). PSCs are GFP‐positive ( green ), the axon terminal is identified by NF staining ( white ). Scale bars: 5 µm. Right panel: the orthogonal projection of one α‐LTx poisoned NMJ shows MT 1 spots along PSC membrane. D, CMAP recordings in gastrocnemius muscles before and 18 d after crush (± luzindole daily i.p. administration, or ±i.p. melatonin, or +luzindole and melatonin). * P < .05, ** P < .01, ns = not significant. F ‐value (DFn, DFd)=10.78 (3,8); P ‐value = .0035. E, MT 1 expression (AMR031, red ) in cross sections of sciatic nerves before (upper panels) and 3 d after crush (lower panels). PSCs are in green (GFP‐positive), axons in white (NF‐positive). Scale bars: 10 µm
    Figure Legend Snippet: MT 1 receptors are expressed at the NMJ and along the sciatic nerve and mediate the pro‐regenerative action of melatonin. A, EJPs amplitude of soleus muscles: (a) in control conditions (± luzindole); (b) +α‐LTx ± i.p. daily treatment with melatonin ± luzindole daily local injections (time point: 72 h after injury); (c) +α‐LTx ± luzindole daily local injections (time point: 96 h after injury). Each bar represents mean ± SEM from N = 4 (number of analyzed fibers: 10). * P < .05, ** P < .01, ns = not significant. F ‐value (DFn, DFd) = 13.65 (4,19); P ‐value < .0001. B, EJPs amplitude of soleus muscles: (a) in control conditions; (b) + α‐LTx ± i.p. daily melatonin; (c) +α‐LTx + i.p. daily melatonin +4P‐PDOT daily local injections; (d) +α‐LTx +ramelteon or tasimelteon daily local injections. For all conditions: time point = 72 h after injury. Each bar represents mean ± SEM from N = 4 (number of analyzed fibers: 10). * P < .05, ** P < .01, *** P < .001, ns = not significant. F ‐value (DFn, DFd) = 9.459 (4,15); P ‐value = .0005. C, MT 1 staining (mAb‐A06, red ) at LAL NMJs in controls (upper panels) and 24 h after α‐LTx injection (lower panels). PSCs are GFP‐positive ( green ), the axon terminal is identified by NF staining ( white ). Scale bars: 5 µm. Right panel: the orthogonal projection of one α‐LTx poisoned NMJ shows MT 1 spots along PSC membrane. D, CMAP recordings in gastrocnemius muscles before and 18 d after crush (± luzindole daily i.p. administration, or ±i.p. melatonin, or +luzindole and melatonin). * P < .05, ** P < .01, ns = not significant. F ‐value (DFn, DFd)=10.78 (3,8); P ‐value = .0035. E, MT 1 expression (AMR031, red ) in cross sections of sciatic nerves before (upper panels) and 3 d after crush (lower panels). PSCs are in green (GFP‐positive), axons in white (NF‐positive). Scale bars: 10 µm

    Techniques Used: Staining, Injection, Expressing

    Melatonin administration prolongs injury‐induced ERK phosphorylation. A, Phospho‐ERK 1/2 signal ( red ) at LAL NMJs in controls and 24/72 h after α‐LTx injection, w/o melatonin i.p. treatment. PSC are green (GFP‐positive). Scale bars: 5 µm. B, Phospho‐ERK 1/2 signal ( red ) in whole‐mount (left) and cross sections (right) of sciatic nerves before and 3 d after crush (w/o melatonin i.p. treatment). White squares indicate the crush site. PSC are in green . Scale bars: 500 µm (left) and 20 µm (right). C, quantification of p‐ERK signal in cross sections. *** P < .001. F ‐value (DFn, DFd) = 123.1 (2,22); P ‐value < .0001
    Figure Legend Snippet: Melatonin administration prolongs injury‐induced ERK phosphorylation. A, Phospho‐ERK 1/2 signal ( red ) at LAL NMJs in controls and 24/72 h after α‐LTx injection, w/o melatonin i.p. treatment. PSC are green (GFP‐positive). Scale bars: 5 µm. B, Phospho‐ERK 1/2 signal ( red ) in whole‐mount (left) and cross sections (right) of sciatic nerves before and 3 d after crush (w/o melatonin i.p. treatment). White squares indicate the crush site. PSC are in green . Scale bars: 500 µm (left) and 20 µm (right). C, quantification of p‐ERK signal in cross sections. *** P < .001. F ‐value (DFn, DFd) = 123.1 (2,22); P ‐value < .0001

    Techniques Used: Injection

    α latrotoxin  (Alomone Labs)


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    Alomone Labs α latrotoxin
    Signal transduction coupled to G protein is altered in mutant ADGRL2 amniocytes. Intracellular calcium levels were monitored by microfluorimetry using the ratiometric Fura-2 AM calcium probe and results expressed as mean fluorescence intensity (MFI). a <t>α-latrotoxin</t> (1 nM) was applied to wild-type (Wt) and mutant (Mt) cultured amniocytes under extracellular chelated-calcium conditions (EDTA 4 mM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. b α-latrotoxin (1 nM) was applied to Wt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. c α-latrotoxin (1 nM) was applied to Mt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. d Quantification and statistical analysis of intracellular calcium levels from the early and late phases in response to α-latrotoxin stimulation. Areas under the curves (AUC) were expressed in arbitrary units (AU). Each value represents the mean (±S.E.M.) of 30 cells. *, p < 0.05; **, p < 0.01, vs Wt amniocytes using one-way ANOVA test
    α Latrotoxin, 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 "A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis"

    Article Title: A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-018-0610-5

    Signal transduction coupled to G protein is altered in mutant ADGRL2 amniocytes. Intracellular calcium levels were monitored by microfluorimetry using the ratiometric Fura-2 AM calcium probe and results expressed as mean fluorescence intensity (MFI). a α-latrotoxin (1 nM) was applied to wild-type (Wt) and mutant (Mt) cultured amniocytes under extracellular chelated-calcium conditions (EDTA 4 mM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. b α-latrotoxin (1 nM) was applied to Wt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. c α-latrotoxin (1 nM) was applied to Mt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. d Quantification and statistical analysis of intracellular calcium levels from the early and late phases in response to α-latrotoxin stimulation. Areas under the curves (AUC) were expressed in arbitrary units (AU). Each value represents the mean (±S.E.M.) of 30 cells. *, p < 0.05; **, p < 0.01, vs Wt amniocytes using one-way ANOVA test
    Figure Legend Snippet: Signal transduction coupled to G protein is altered in mutant ADGRL2 amniocytes. Intracellular calcium levels were monitored by microfluorimetry using the ratiometric Fura-2 AM calcium probe and results expressed as mean fluorescence intensity (MFI). a α-latrotoxin (1 nM) was applied to wild-type (Wt) and mutant (Mt) cultured amniocytes under extracellular chelated-calcium conditions (EDTA 4 mM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. b α-latrotoxin (1 nM) was applied to Wt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. c α-latrotoxin (1 nM) was applied to Mt amniocytes under chelated-calcium conditions (EDTA 4 mM). Cultured cells were pre-incubated or not with the phospholipase C inhibitor U73122 (10 μM). Three minutes after α-latrotoxin administration, cells were perfused without EDTA to restore extracellular calcium levels. d Quantification and statistical analysis of intracellular calcium levels from the early and late phases in response to α-latrotoxin stimulation. Areas under the curves (AUC) were expressed in arbitrary units (AU). Each value represents the mean (±S.E.M.) of 30 cells. *, p < 0.05; **, p < 0.01, vs Wt amniocytes using one-way ANOVA test

    Techniques Used: Transduction, Mutagenesis, Fluorescence, Cell Culture, Incubation

    HeLa cells overexpressing mutant ADGRL2 present enhanced cell adhesive properties associated to signal transduction alteration. a-c HeLa cells expressing either pcD-Empty ( a ), CIRL2-Wt ( b ) or CIRL2-Mt ( c ) were labelled with the viability marker, cell tracker green (10 μM), and the mortality marker 7-AAD (50 μg/ml) and incubated at room temperature for 90 min in aggregation medium. Note the marked increase of aggregate sizes in CIRL2-Mt expressing cells. d-f HeLa cells expressing either pcD-Empty ( d ) CIRL2-Wt ( e ) or CIRL2-Mt ( f ) were incubated at room temperature for 90 min in aggregation medium containing the PLC inhibitor U73122 (3 μM). Note that inhibition of PLC enhanced homophilic binding of HeLa cells overexpressing CIRL2-Wt. g-i HeLa cells expressing either pcD-Empty ( g ), CIRL2-Wt ( h ) or CIRL2-Mt ( i ) were incubated at room temperature for 90 min in aggregation medium containing α-latrotoxin (1 nM) which prevented cell aggregation. j-l HeLa cells expressing either pcD-GFP-Empty ( j ), CIRL2-GFP-Wt ( k ) or CRL2-GFP-Mt ( l ) were incubated at room temperature for 90 min in aggregation medium. Cells expressing CIRL2 coupled to GFP in C-terminal were not able to aggregate. m Quantification and statistical analysis of the aggregation index. Each value represents the mean (±S.E.M.) of three independent cell-adhesion assays. **, p < 0.01; ***, p < 0.001, ****, p < 0.0001 using one-way ANOVA test
    Figure Legend Snippet: HeLa cells overexpressing mutant ADGRL2 present enhanced cell adhesive properties associated to signal transduction alteration. a-c HeLa cells expressing either pcD-Empty ( a ), CIRL2-Wt ( b ) or CIRL2-Mt ( c ) were labelled with the viability marker, cell tracker green (10 μM), and the mortality marker 7-AAD (50 μg/ml) and incubated at room temperature for 90 min in aggregation medium. Note the marked increase of aggregate sizes in CIRL2-Mt expressing cells. d-f HeLa cells expressing either pcD-Empty ( d ) CIRL2-Wt ( e ) or CIRL2-Mt ( f ) were incubated at room temperature for 90 min in aggregation medium containing the PLC inhibitor U73122 (3 μM). Note that inhibition of PLC enhanced homophilic binding of HeLa cells overexpressing CIRL2-Wt. g-i HeLa cells expressing either pcD-Empty ( g ), CIRL2-Wt ( h ) or CIRL2-Mt ( i ) were incubated at room temperature for 90 min in aggregation medium containing α-latrotoxin (1 nM) which prevented cell aggregation. j-l HeLa cells expressing either pcD-GFP-Empty ( j ), CIRL2-GFP-Wt ( k ) or CRL2-GFP-Mt ( l ) were incubated at room temperature for 90 min in aggregation medium. Cells expressing CIRL2 coupled to GFP in C-terminal were not able to aggregate. m Quantification and statistical analysis of the aggregation index. Each value represents the mean (±S.E.M.) of three independent cell-adhesion assays. **, p < 0.01; ***, p < 0.001, ****, p < 0.0001 using one-way ANOVA test

    Techniques Used: Mutagenesis, Transduction, Expressing, Marker, Incubation, Inhibition, Binding Assay

    alphalatrotoxin  (Alomone Labs)


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    Alomone Labs alphalatrotoxin
    Alphalatrotoxin, 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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    alpha latrotoxin  (Alomone Labs)


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    Alomone Labs alpha latrotoxin
    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of <t>α‐latrotoxin</t> (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.
    Alpha Latrotoxin, 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 "Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse"

    Article Title: Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse

    Journal: The Journal of Physiology

    doi: 10.1113/JP275107

    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.
    Figure Legend Snippet: A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.

    Techniques Used: Fluorescence

    alpha latrotoxin  (Alomone Labs)


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    Alomone Labs alpha latrotoxin
    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of <t>α‐latrotoxin</t> (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.
    Alpha Latrotoxin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse"

    Article Title: Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse

    Journal: The Journal of Physiology

    doi: 10.1113/JP275107

    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.
    Figure Legend Snippet: A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.

    Techniques Used: Fluorescence

    α latrotoxin  (Alomone Labs)


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    Alomone Labs α latrotoxin
    Synaptic vesicle exocytosis is associated with α-synuclein release. a Schematic illustration of target molecules for the compounds used in the experiment. b <t>α-Latrotoxin</t> (αLTX, 0.5 nM) increased α-synuclein release, which was blocked by 4 mM EGTA. N = 8. c αLTX significantly increased α-synuclein release in the presence or absence of 1 μM TTX, 50 μM AP5 and 10 μM NBQX. N = 16–32. d LDH activities in the media were not altered by indicated pharmacological treatments. N = 16–24. e Cellular α-synuclein levels were not altered by indicate pharmacological treatments. N = 8. mean ± SEM, *** p < 0.001, **** p < 0.0001 using one-way ANOVA
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    1) Product Images from "Extracellular α-synuclein levels are regulated by neuronal activity"

    Article Title: Extracellular α-synuclein levels are regulated by neuronal activity

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-018-0241-0

    Synaptic vesicle exocytosis is associated with α-synuclein release. a Schematic illustration of target molecules for the compounds used in the experiment. b α-Latrotoxin (αLTX, 0.5 nM) increased α-synuclein release, which was blocked by 4 mM EGTA. N = 8. c αLTX significantly increased α-synuclein release in the presence or absence of 1 μM TTX, 50 μM AP5 and 10 μM NBQX. N = 16–32. d LDH activities in the media were not altered by indicated pharmacological treatments. N = 16–24. e Cellular α-synuclein levels were not altered by indicate pharmacological treatments. N = 8. mean ± SEM, *** p < 0.001, **** p < 0.0001 using one-way ANOVA
    Figure Legend Snippet: Synaptic vesicle exocytosis is associated with α-synuclein release. a Schematic illustration of target molecules for the compounds used in the experiment. b α-Latrotoxin (αLTX, 0.5 nM) increased α-synuclein release, which was blocked by 4 mM EGTA. N = 8. c αLTX significantly increased α-synuclein release in the presence or absence of 1 μM TTX, 50 μM AP5 and 10 μM NBQX. N = 16–32. d LDH activities in the media were not altered by indicated pharmacological treatments. N = 16–24. e Cellular α-synuclein levels were not altered by indicate pharmacological treatments. N = 8. mean ± SEM, *** p < 0.001, **** p < 0.0001 using one-way ANOVA

    Techniques Used:

    α ltx  (Alomone Labs)


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    Structured Review

    Alomone Labs α ltx
    A, B The time course of motor axon terminal degeneration and regeneration induced by <t>α‐LTx</t> at LAL NMJs was determined in mice with GFP‐expressing SCs (green), using the presynaptic markers SNAP25 (red) (A) and VAMP1 (red) (B); at 4 h, SNAP25 and VAMP1 are phagocytosed inside PSCs. Muscles were fixed 0, 4, 24, and 96 h post‐injection. Scale bars: 10 μm. C CXCL12α mRNA levels (expressed as FPKM, fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α‐LTx at LAL NMJs. Data are presented as mean ± SD. * P = 0.017 (ctr vs. 4 h), three independent experiments. Statistical analysis was performed by Cuffdiff software. D CXCL12α mRNA levels measured by droplet digital PCR performed on cDNA from soleus muscles locally injected with α‐LTx (0, 4, 16, and 72 h). The time points analyzed were chosen on the basis of the time course of nerve terminal degeneration and regeneration by α‐LTx poisoning in soleus muscle, which is slightly different from that of LAL. Nonetheless, in both muscles degeneration peaks at 4 h (Fig ). Data are expressed as fractionary abundance with respect to the housekeeping GAPDH, five independent experiments. Data are presented as mean ± SD. **** P < 0.0001 (4 h vs. ctr, 16 and 72 h) by ANOVA with post hoc Tukey test. E In situ CXCL12α mRNA hybridization (white) at soleus NMJ before and after 4‐h intoxication with α‐LTx. PSCs are in green. Representative images are shown. Scale bars: 10 μm. F Immunostaining for CXCL12α (white, arrows) at LAL NMJs in controls and after 4, 24, and 96 h of intoxication. PSCs are in green. Scale bars: 10 μm. Right: Orthogonal projection of α‐LTx‐poisoned NMJ (4 h) shows that CXCL12α spots are inside PSCs (arrows). Scale bar: 10 μm.
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    Images

    1) Product Images from "CXCL 12α/ SDF ‐1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals"

    Article Title: CXCL 12α/ SDF ‐1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201607257

    A, B The time course of motor axon terminal degeneration and regeneration induced by α‐LTx at LAL NMJs was determined in mice with GFP‐expressing SCs (green), using the presynaptic markers SNAP25 (red) (A) and VAMP1 (red) (B); at 4 h, SNAP25 and VAMP1 are phagocytosed inside PSCs. Muscles were fixed 0, 4, 24, and 96 h post‐injection. Scale bars: 10 μm. C CXCL12α mRNA levels (expressed as FPKM, fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α‐LTx at LAL NMJs. Data are presented as mean ± SD. * P = 0.017 (ctr vs. 4 h), three independent experiments. Statistical analysis was performed by Cuffdiff software. D CXCL12α mRNA levels measured by droplet digital PCR performed on cDNA from soleus muscles locally injected with α‐LTx (0, 4, 16, and 72 h). The time points analyzed were chosen on the basis of the time course of nerve terminal degeneration and regeneration by α‐LTx poisoning in soleus muscle, which is slightly different from that of LAL. Nonetheless, in both muscles degeneration peaks at 4 h (Fig ). Data are expressed as fractionary abundance with respect to the housekeeping GAPDH, five independent experiments. Data are presented as mean ± SD. **** P < 0.0001 (4 h vs. ctr, 16 and 72 h) by ANOVA with post hoc Tukey test. E In situ CXCL12α mRNA hybridization (white) at soleus NMJ before and after 4‐h intoxication with α‐LTx. PSCs are in green. Representative images are shown. Scale bars: 10 μm. F Immunostaining for CXCL12α (white, arrows) at LAL NMJs in controls and after 4, 24, and 96 h of intoxication. PSCs are in green. Scale bars: 10 μm. Right: Orthogonal projection of α‐LTx‐poisoned NMJ (4 h) shows that CXCL12α spots are inside PSCs (arrows). Scale bar: 10 μm.
    Figure Legend Snippet: A, B The time course of motor axon terminal degeneration and regeneration induced by α‐LTx at LAL NMJs was determined in mice with GFP‐expressing SCs (green), using the presynaptic markers SNAP25 (red) (A) and VAMP1 (red) (B); at 4 h, SNAP25 and VAMP1 are phagocytosed inside PSCs. Muscles were fixed 0, 4, 24, and 96 h post‐injection. Scale bars: 10 μm. C CXCL12α mRNA levels (expressed as FPKM, fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α‐LTx at LAL NMJs. Data are presented as mean ± SD. * P = 0.017 (ctr vs. 4 h), three independent experiments. Statistical analysis was performed by Cuffdiff software. D CXCL12α mRNA levels measured by droplet digital PCR performed on cDNA from soleus muscles locally injected with α‐LTx (0, 4, 16, and 72 h). The time points analyzed were chosen on the basis of the time course of nerve terminal degeneration and regeneration by α‐LTx poisoning in soleus muscle, which is slightly different from that of LAL. Nonetheless, in both muscles degeneration peaks at 4 h (Fig ). Data are expressed as fractionary abundance with respect to the housekeeping GAPDH, five independent experiments. Data are presented as mean ± SD. **** P < 0.0001 (4 h vs. ctr, 16 and 72 h) by ANOVA with post hoc Tukey test. E In situ CXCL12α mRNA hybridization (white) at soleus NMJ before and after 4‐h intoxication with α‐LTx. PSCs are in green. Representative images are shown. Scale bars: 10 μm. F Immunostaining for CXCL12α (white, arrows) at LAL NMJs in controls and after 4, 24, and 96 h of intoxication. PSCs are in green. Scale bars: 10 μm. Right: Orthogonal projection of α‐LTx‐poisoned NMJ (4 h) shows that CXCL12α spots are inside PSCs (arrows). Scale bar: 10 μm.

    Techniques Used: Expressing, Injection, Software, Digital PCR, In Situ, Hybridization, Immunostaining

    Mice were injected with α‐LTx in proximity to LAL and fixed at different time points during MAT degeneration and regeneration, defined on the basis of the kinetics reported in Fig A and B. After muscle collection, 50 NMJs/sample were laser‐microdissected, pooled, and processed for NGS (next‐generation sequencing).
    Figure Legend Snippet: Mice were injected with α‐LTx in proximity to LAL and fixed at different time points during MAT degeneration and regeneration, defined on the basis of the kinetics reported in Fig A and B. After muscle collection, 50 NMJs/sample were laser‐microdissected, pooled, and processed for NGS (next‐generation sequencing).

    Techniques Used: Injection, Next-Generation Sequencing

    The time course of MAT degeneration and regeneration induced by α‐LTx at soleus NMJs was determined in mice with GFP‐expressing SCs (green), using the presynaptic marker VAMP1 (red). The post‐synaptic differentiations are identified by α‐bungarotoxin (α‐BTx) staining (white). Muscles were fixed 0, 4, 16, and 72 h post‐injection. Scale bars: 10 μm.
    Figure Legend Snippet: The time course of MAT degeneration and regeneration induced by α‐LTx at soleus NMJs was determined in mice with GFP‐expressing SCs (green), using the presynaptic marker VAMP1 (red). The post‐synaptic differentiations are identified by α‐bungarotoxin (α‐BTx) staining (white). Muscles were fixed 0, 4, 16, and 72 h post‐injection. Scale bars: 10 μm.

    Techniques Used: Expressing, Marker, Staining, Injection

    A Evoked junctional potentials (EJPs) recorded in soleus muscles 48, 72 and 96 h after α‐LTx injection in the mice hind limb, with/without a previous intraperitoneal administration of a CXCL12α‐neutralizing antibody. Controlateral muscles (injected with saline) were used as controls. Muscles injected with the sole neutralizing antibody show EJPs similar to controls. Each bar represents mean ± SEM from six animals, 15 EJPs measured per animal. * P = 0.03 (72 h) and * P = 0.017 (96 h) by Student's t‐ test, unpaired, two‐sided. B, C CXCL12α promotes axon growth of primary SCMNs. r CXCL12α (500 ng/ml) was added to SCMNs plated in culture dishes. After 24 h, neurons were fixed and stained for β 3 ‐tubulin (green). Scale bar: 10 μm. Quantification is shown in (C). Each bar represents the mean ± SD from five different experiments, 70 neurons measured per experiment. *** P = 0.0003 by Student's t‐ test, unpaired, two‐sided. D Microfluidic devices employed in the study. Somatic chambers are separated from the distal ones by a series of grooves along which SCMNs axons grow (cells plated in the somatic compartment). E, F r CXCL12α (500 ng/ml) added to the distal chamber promotes axonal elongation of SCMNs plated in the somatic chamber of microfluidic devices. The figure shows the distal chamber after 5 days of culture. Scale bars: 50 μm. Panel (F) shows the quantification of axon growth from nine experiments. Data are presented as mean ± SD. ** P = 0.0019 by Student's t‐ test, unpaired, two‐sided. G Evoked junctional potentials (EJPs) recorded in soleus muscles 72 h after α‐LTx injection in the mice hind limb, with/without local administrations of r CXCL12α. Controlateral muscles were used as controls. Each bar represents mean ± SEM from nine animals, 15 EJPs measured per animal. ** P = 0.0043 by Student's t‐ test, unpaired, two‐sided.
    Figure Legend Snippet: A Evoked junctional potentials (EJPs) recorded in soleus muscles 48, 72 and 96 h after α‐LTx injection in the mice hind limb, with/without a previous intraperitoneal administration of a CXCL12α‐neutralizing antibody. Controlateral muscles (injected with saline) were used as controls. Muscles injected with the sole neutralizing antibody show EJPs similar to controls. Each bar represents mean ± SEM from six animals, 15 EJPs measured per animal. * P = 0.03 (72 h) and * P = 0.017 (96 h) by Student's t‐ test, unpaired, two‐sided. B, C CXCL12α promotes axon growth of primary SCMNs. r CXCL12α (500 ng/ml) was added to SCMNs plated in culture dishes. After 24 h, neurons were fixed and stained for β 3 ‐tubulin (green). Scale bar: 10 μm. Quantification is shown in (C). Each bar represents the mean ± SD from five different experiments, 70 neurons measured per experiment. *** P = 0.0003 by Student's t‐ test, unpaired, two‐sided. D Microfluidic devices employed in the study. Somatic chambers are separated from the distal ones by a series of grooves along which SCMNs axons grow (cells plated in the somatic compartment). E, F r CXCL12α (500 ng/ml) added to the distal chamber promotes axonal elongation of SCMNs plated in the somatic chamber of microfluidic devices. The figure shows the distal chamber after 5 days of culture. Scale bars: 50 μm. Panel (F) shows the quantification of axon growth from nine experiments. Data are presented as mean ± SD. ** P = 0.0019 by Student's t‐ test, unpaired, two‐sided. G Evoked junctional potentials (EJPs) recorded in soleus muscles 72 h after α‐LTx injection in the mice hind limb, with/without local administrations of r CXCL12α. Controlateral muscles were used as controls. Each bar represents mean ± SEM from nine animals, 15 EJPs measured per animal. ** P = 0.0043 by Student's t‐ test, unpaired, two‐sided.

    Techniques Used: Injection, Staining

    A, B Anatomical recovery of soleus NMJs 72 h after α‐LTx injection in the mice hind limb, with/without a previous intraperitoneal administration of a CXCL12α‐neutralizing antibody, as assessed by immunostaining the presynaptic marker VAMP1 (red). GFP‐expressing SCs are in green. The post‐synaptic differentiations are identified by α‐bungarotoxin (α‐BTx) staining (white). Asterisks identify those NMJs that are still degenerated (positive for α‐BTx but negative for VAMP1). Scale bars: 10 μm. Quantitation is shown in (B). Data are presented as mean ± SD. * P = 0.0123 by Student's t‐ test, unpaired, two‐sided.
    Figure Legend Snippet: A, B Anatomical recovery of soleus NMJs 72 h after α‐LTx injection in the mice hind limb, with/without a previous intraperitoneal administration of a CXCL12α‐neutralizing antibody, as assessed by immunostaining the presynaptic marker VAMP1 (red). GFP‐expressing SCs are in green. The post‐synaptic differentiations are identified by α‐bungarotoxin (α‐BTx) staining (white). Asterisks identify those NMJs that are still degenerated (positive for α‐BTx but negative for VAMP1). Scale bars: 10 μm. Quantitation is shown in (B). Data are presented as mean ± SD. * P = 0.0123 by Student's t‐ test, unpaired, two‐sided.

    Techniques Used: Injection, Immunostaining, Marker, Expressing, Staining, Quantitation Assay

    A, B CXCR4 (red) is expressed by primary SCMNs (β 3 ‐tubulin positive, green) and accumulates in growing tips (arrows). Panel (B) shows CXCR4 co‐localization with the growth‐cone marker GAP43 (green). Scale bars: 10 μm. C CXCR4 (white) is almost undetectable in control NMJs from LAL muscles, and it becomes evident in nerve terminals (SNAP25‐NF positive, red) poisoned by α‐LTx (16‐h intoxication). GFP‐expressing SCs are in green. Scale bars: 10 μm. D, E AMD3100 reduces the axon elongation stimulating ability of r CXCL12α on SCMNs in microfluidic chambers. Axon growth was measured after 5 days of treatment. Scale bars: 10 μm. Quantification is shown in (E). Each bar represents mean ± SD from six different experiments, 70 neurons measured per experiment. **** P < 0.0001 by ANOVA followed by post hoc Tukey test. F Intraperitoneal administration of the CXCR4 antagonist AMD3100 delays the functional recovery of mice NMJs exposed to α‐LTx. Each bar represents mean ± SEM from six animals, 15 EJPs measured per animal. ** P = 0.0057 by Student's t‐ test, unpaired, two‐sided.
    Figure Legend Snippet: A, B CXCR4 (red) is expressed by primary SCMNs (β 3 ‐tubulin positive, green) and accumulates in growing tips (arrows). Panel (B) shows CXCR4 co‐localization with the growth‐cone marker GAP43 (green). Scale bars: 10 μm. C CXCR4 (white) is almost undetectable in control NMJs from LAL muscles, and it becomes evident in nerve terminals (SNAP25‐NF positive, red) poisoned by α‐LTx (16‐h intoxication). GFP‐expressing SCs are in green. Scale bars: 10 μm. D, E AMD3100 reduces the axon elongation stimulating ability of r CXCL12α on SCMNs in microfluidic chambers. Axon growth was measured after 5 days of treatment. Scale bars: 10 μm. Quantification is shown in (E). Each bar represents mean ± SD from six different experiments, 70 neurons measured per experiment. **** P < 0.0001 by ANOVA followed by post hoc Tukey test. F Intraperitoneal administration of the CXCR4 antagonist AMD3100 delays the functional recovery of mice NMJs exposed to α‐LTx. Each bar represents mean ± SEM from six animals, 15 EJPs measured per animal. ** P = 0.0057 by Student's t‐ test, unpaired, two‐sided.

    Techniques Used: Marker, Expressing, Functional Assay

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    Alomone Labs α latrotoxin
    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM <t>α-latrotoxin</t> (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.
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    Alomone Labs α ltx
    Time course of NMJ degeneration and regeneration by <t>α-LTx</t> reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.
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    Alomone Labs alphalatrotoxin
    Time course of NMJ degeneration and regeneration by <t>α-LTx</t> reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.
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    Alomone Labs alpha latrotoxin
    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of <t>α‐latrotoxin</t> (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.
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    Image Search Results


    Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.

    Journal: Neural Development

    Article Title: Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

    doi: 10.1186/1749-8104-3-25

    Figure Lengend Snippet: Electrophysiological assessment shows normal synaptic transmission at transforming growth factor (TGF)-β2 knock-out (KO) neuromuscular junctions (NMJs). (a-h) Bar graphs display the group mean values ± standard error of the mean (n = 3 and 5 embryos for the TGF-β2 KO and control (CON) groups, respectively; data from 3–20 NMJs sampled per muscle). (a) Amplitude, (b) rise-time and (c) frequency of miniature endplate potentials (MEPPs) are unchanged ( P = 0.52, 0.23 and 0.11, respectively). (d) MEPP frequency evoked by 2.5 nM α-latrotoxin (α-LTx) is normal ( P = 0.48). (e) Similar delay between nerve stimulation and start of the recorded postsynaptic response, either an action potential or an endplate potential (EPP; P = 0.49). (f) Rise time and (g) amplitude of EPPs are normal, as well as (h) the calculated quantal content, that is, the number of transmitter quanta released upon one nerve impulse. (i) Example traces of MEPP recordings in normal Ringer's medium (left panel, showing all MEPPs encountered during a 5 minute recording period) and in the presence of 2.5 nM α-LTx (right panel, 1 s recorded). (j) Examples of recorded muscle fiber action potentials following from a single nerve stimulation. The ensuing contraction of the impaled fiber (and neighboring fibers) leads to the contraction artifact visible in the recording trace just after the action potential. (k) At fibers that were allowed to depolarize to around -30 mV by waiting for some time, a single nerve stimulation evoked an EPP. Example traces of EPPs with similar characteristics recorded in TGF-β2 KO and control fibers. Contraction of neighboring fibers is visible as an artifact on the signal, starting just after the EPP.

    Article Snippet: MEPPs were also recorded after application of 2.5 nM α-latrotoxin (Alomone Laboratories, Jerusalem, Israel).

    Techniques: Transmission Assay, Knock-Out

    Time course of NMJ degeneration and regeneration by α-LTx reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.

    Journal: International Journal of Molecular Sciences

    Article Title: Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration

    doi: 10.3390/ijms23031186

    Figure Lengend Snippet: Time course of NMJ degeneration and regeneration by α-LTx reveals a strong upregulation of the UCN2 gene. ( a ) Time course of MAT degeneration and regeneration induced by α-LTx in soleus muscles assessed by immunostaining at 4, 16, 72, 168 h after intoxication. SCs (green) are GFP-positive, SNAP25 (red) identifies the presynaptic nerve terminals. Scale bars = 10 μm. ( b ) Time course of loss and progressive recovery of motor axon activity by evoked EPP recordings in soleus muscles at the indicated time points. Histograms show the average amplitude of evoked EPPs ± SD. n = 4 animals, 11 fibres each. **** p < 0.0001. ANOVA followed by post hoc Tukey test. ( c ) Quantification of regenerated NMJs in soleus muscles at the indicated time points after α-LTx injection. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 and nAChR stained with α-BTx, respectively). Each bar represents the mean ± SD percentage of recovered NMJs compared to non-treated animals. **** p < 0.0001, ** p < 0.01 by ANOVA followed by post hoc Tukey test. ( d ) Experimental pipeline of NMJ transcriptome profiling throughout the process of degeneration and regeneration caused by α-LTx. Mice were injected with the toxin close to LAL muscle, which was then isolated and snap-frozen at the indicated time points. Single NMJs were isolated by LCM from cryo-sliced muscles. At least 150 NMJs were pooled and their RNA extracted, amplified and sequenced with the Ion Torrent technology. ( e ) Scheme depicting the workflow and criteria used to process the NMJ dataset. ( f ) Graph showing UCN2 mRNA profile, expressed as FPKM (fragments per kilobase of exon per million fragments mapped) during MAT degeneration and regeneration induced by α-LTx in LAL muscles. Data are presented as mean ± SD. **** p < 0.0001 (ctr vs. 24 h) from 3 independent replicates. ( g ) Experimental workflow to assess UCN2 mRNA levels in soleus muscles 4, 16, 72, 168 h after local injection of α-LTx. Time points were chosen on the basis of the time course of MAT degeneration and regeneration by α-LTx as described above. ( h ) UCN2 mRNA levels measured via digital PCR on NMJs isolated by LCM from soleus muscles. Data are expressed as fold change compared to control. Each bar represents mean ± SD. n = 4 animals. **** p < 0.0001, * p < 0.05. ANOVA followed by post hoc Tukey test.

    Article Snippet: Fluorescent Mounting Medium was purchased from Dako Agilent (Santa Clara, CA, USA). µ-Conotoxin GIIIB (cat.C-270) and α-LTx (cat. LSP-130) were purchased from Alomone (Jerusalem, Israel).

    Techniques: Immunostaining, Activity Assay, Injection, Staining, Isolation, Amplification, Digital PCR

    The CRHR2 receptor participates in the structural and functional recovery of the NMJ. ( a ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or the CRHR2 antagonist A2B up to 96 h after α-LTx injection, and the analysis performed to assess the functional and structural status of the NMJ. ( b ) Electrophysiological recordings of evoked EPPs from intoxicated soleus muscles after either vehicle of A2B administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. ** p < 0.01, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( c ) Representative immunostaining and ( d ) quantification performed on soleus muscles after intoxication with α-LTx and treatment with either vehicle or A2B. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (lack or little VAMP-1 signal). The right panel shows a magnification. Scale bars: 50 µm. Bars in ( d ) represent the mean± SD percentage of regenerated NMJs. ** p < 0.01 by Student’s t -test.

    Journal: International Journal of Molecular Sciences

    Article Title: Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration

    doi: 10.3390/ijms23031186

    Figure Lengend Snippet: The CRHR2 receptor participates in the structural and functional recovery of the NMJ. ( a ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or the CRHR2 antagonist A2B up to 96 h after α-LTx injection, and the analysis performed to assess the functional and structural status of the NMJ. ( b ) Electrophysiological recordings of evoked EPPs from intoxicated soleus muscles after either vehicle of A2B administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. ** p < 0.01, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( c ) Representative immunostaining and ( d ) quantification performed on soleus muscles after intoxication with α-LTx and treatment with either vehicle or A2B. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (lack or little VAMP-1 signal). The right panel shows a magnification. Scale bars: 50 µm. Bars in ( d ) represent the mean± SD percentage of regenerated NMJs. ** p < 0.01 by Student’s t -test.

    Article Snippet: Fluorescent Mounting Medium was purchased from Dako Agilent (Santa Clara, CA, USA). µ-Conotoxin GIIIB (cat.C-270) and α-LTx (cat. LSP-130) were purchased from Alomone (Jerusalem, Israel).

    Techniques: Functional Assay, Injection, Immunostaining

    The UCN2-CRHR2 axis promotes axonal growth of cultured SCMNs and NMJ regeneration after acute damage. ( a ) Representative images of neurofilament staining used as reporter for axon length in SCMNs cultured for 24 h either in normal culture medium (NC) or NC supplemented with UCN2, A2B or their combination. ( b ) Quantification of the average axonal length after the indicated treatments. Three independent experiments were performed, and at least 100 neurons per condition measured. Bars represent mean ± SD. Statical analysis was performed with ANOVA followed by post hoc Tukey test; *** p < 0.001, ** p < 0.01, ns p > 0.05. ( c ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or recombinant UCN2 for 72 h after α-LTx treatment, followed by functional and structural assessment of NMJ status. ( d ) Electrophysiological assessment of NMJ function by evoked EPPs recordings following either vehicle or UCN2 administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. * p < 0.05, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( e ) Representative immunostaining and ( f ) quantification performed on soleus muscles after intoxication and treatment with either vehicle or UCN2. Regenerated NMJs are positive for both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (absent or faint VAMP1 signal). Right panels are magnifications of the indicated NMJs. Scale bars: 50 µm, 20 µm for magnification. Each bar in ( f ) represents the mean ± SD percentage of recovered NMJs. ** p < 0.01 by Student’s t -test.

    Journal: International Journal of Molecular Sciences

    Article Title: Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration

    doi: 10.3390/ijms23031186

    Figure Lengend Snippet: The UCN2-CRHR2 axis promotes axonal growth of cultured SCMNs and NMJ regeneration after acute damage. ( a ) Representative images of neurofilament staining used as reporter for axon length in SCMNs cultured for 24 h either in normal culture medium (NC) or NC supplemented with UCN2, A2B or their combination. ( b ) Quantification of the average axonal length after the indicated treatments. Three independent experiments were performed, and at least 100 neurons per condition measured. Bars represent mean ± SD. Statical analysis was performed with ANOVA followed by post hoc Tukey test; *** p < 0.001, ** p < 0.01, ns p > 0.05. ( c ) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or recombinant UCN2 for 72 h after α-LTx treatment, followed by functional and structural assessment of NMJ status. ( d ) Electrophysiological assessment of NMJ function by evoked EPPs recordings following either vehicle or UCN2 administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. * p < 0.05, ns p > 0.05. ANOVA followed by post hoc Tukey test. ( e ) Representative immunostaining and ( f ) quantification performed on soleus muscles after intoxication and treatment with either vehicle or UCN2. Regenerated NMJs are positive for both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (absent or faint VAMP1 signal). Right panels are magnifications of the indicated NMJs. Scale bars: 50 µm, 20 µm for magnification. Each bar in ( f ) represents the mean ± SD percentage of recovered NMJs. ** p < 0.01 by Student’s t -test.

    Article Snippet: Fluorescent Mounting Medium was purchased from Dako Agilent (Santa Clara, CA, USA). µ-Conotoxin GIIIB (cat.C-270) and α-LTx (cat. LSP-130) were purchased from Alomone (Jerusalem, Israel).

    Techniques: Cell Culture, Staining, Recombinant, Functional Assay, Immunostaining

    A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.

    Journal: The Journal of Physiology

    Article Title: Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse

    doi: 10.1113/JP275107

    Figure Lengend Snippet: A, representative images of calyx of Held synapses labelled with FM1‐43FX; labelling of vesicles in the presynaptic calyx is shown in green surrounding a central unstained (black) MNTB neuron. Left column shows 10 mm glucose the right column shows zero glucose before HFS (top), after HFS (middle) and after 2 min application of α‐latrotoxin (bottom). Scale bar 10 μm. B, summary graph of FM1‐43FX fluorescence intensity plotted over time during the 25 min HFS (black bar) for calyces from slices perfused with 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12). Inset: ratios of fluorescence intensity after Lat application over initial FM‐dye fluorescence for calyces perfused in 10 mm glucose (grey, n = 14) and zero glucose (black, n = 12) show reduced vesicle recycling in the zero glucose condition.

    Article Snippet: Other drugs were purchased as listed here: monocarboxylate transporter inhibitor AR‐C155858 (Tocris, Bristol, UK; 4960), FM1‐43FX (Molecular Probes, Carlsbad, CA, USA; cat. no. {"type":"entrez-nucleotide","attrs":{"text":"F35355","term_id":"4820981","term_text":"F35355"}} F35355 ), alpha‐Latrotoxin (Alomone, Jerusalem, Israel; LSP‐130), Bromophenol Blue (Acros, cat. no. 151340250).

    Techniques: Fluorescence