primary antibody rabbit-anti-ca v 2.1 #152203  (Synaptic Systems)

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic illustrating imaging at the plasma membrane (PM). B Left: representative single-plane Airyscan confocal images of the PM showing Ca V 1.2 immunolabeling in control (CTL, black) and PFF-treated (red) neurons. Inset: MAP2 (pink) neuronal marker. Right: quantification of Ca V 1.2 cluster size, cluster density, and mean gray value (MGV) in the soma (a.) and dendrites (b.) of CTL (black) and PFF-treated (red) neurons. Dendritic measurements are shown separately for excitatory (dark blue) and inhibitory (light blue) populations. n = 20 somata per condition; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations with each isolation containing 8-10 pups. C Left: representative super-resolution TIRF localization maps showing Ca V 1.2 immunolabeling in CTL (black) and PFF-treated (red) neurons. Right: quantification of PM Ca V 1.2 cluster size, cluster density, and nearest-neighbor distance in the somatic region. n = 16 neurons per condition; two independent isolations. D Same experimental design as in ( B ), with neurons immunolabeled for Ca V 2.1. n = 19 (CTL) and n = 20 (PFF) somata; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations. E Same experimental design as in ( C ), with neurons immunolabeled for Ca V 2.1. n = 16 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance was determined using two-tailed Mann-Whitney or unpaired two-tailed t-tests. ns, not significant; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Imaging, Clinical Proteomics, Membrane, Immunolabeling, Control, Marker, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic representation of the CCAD peptide mechanism of action. B Left: representative single-plane Airyscan confocal images of the PM in CTL and PFF-treated neurons co-incubated with SCRBL or CCAD peptides and co-immunolabeled for Ca V 1.2 and K V 2.1. Conditions are shown as CTL;SCRBL (black), PFF;SCRBL (red), CTL;CCAD (gray), and PFF;CCAD (yellow). Right: quantification of Ca V 1.2 cluster size, K V 2.1 cluster size, and Ca V 1.2-K V 2.1 overlap area in the somatic region. n = 19 (CTL;SCRBL), n = 19 (PFF;SCRBL), n = 20 (CTL;CCAD), and n = 20 (PFF;CCAD) neurons; two independent isolations. C Schematic representation of the proximity ligation assay (PLA). D Top: representative Airyscan confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( B ). Bottom: quantification of PLA puncta density. n = 18 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 18 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance in panels ( B, D-E ) was determined using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; CCAD, calcium channel association domain peptide; SCRBL, scrambled control peptide.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Incubation, Immunolabeling, Proximity Ligation Assay, Control

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Left: representative FV4000 confocal images showing co-immunolabeling of CDK5 and K V 2.1 at the PM in control (CTL, black) and PFF-treated (red) neurons. Images are maximum intensity projections from three optical sections acquired at the PM. Right: quantification of CDK5-K V 2.1 overlap area and CDK5 puncta density in the somatic region. n = 18 (CTL) and n = 19 (PFF) neurons; two independent isolations. B Schematic representation of the roscovitine mechanism of action. C Top: representative FV4000 confocal images of the PM in CTL and PFF-treated neurons incubated with or without roscovitine and immunolabeled for pS603-K V 2.1. Conditions are shown as CTL (black), PFF (red), CTL;Rosco (blue), and PFF;Rosco (purple). Images are maximum intensity projections from Z-stacks spanning whole cells. Bottom: quantification of somatic pS603-Kv2.1 occupancy (% of soma area), cluster MGV, and cluster density. n = 19 (CTL), n = 20 (PFF), n = 20 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. D Left: representative FV4000 confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( C ). Right: quantification of PLA puncta density. n = 20 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 20 (CTL), n = 20 (PFF), n = 21 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. Error bars represent SEM. Statistical significance in panel ( A ) was determined using two-tailed Mann-Whitney test; panels ( C – E ) were analyzed using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; Rosco, roscovitine; pS603-K V 2.1, K V 2.1 phosphorylated at serine 603.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Immunolabeling, Control, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Pain

Article Title: Familial hemiplegic migraine Ca V 2.1 channel mutation R192Q enhances ATP-gated P2X 3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

doi: 10.1186/1744-8069-6-48

Figure Lengend Snippet: Ca 2+ transients evoked by K + depolarization or P2X 3 receptors in WT and R192Q KI neurons . A , Examples of Ca 2+ transients of trigeminal neurons evoked by KCl (20 mM, 2-s application) before ( black trace ) and after ( red trace ) application of ω-agatoxin (200 nM, 30 min). B , KI neurons show significant increase in KCl (20 mM, 2-s application) mediated Ca 2+ transients compared to WT (* p = 0.005, n = 28 and n = 45, in WT and KI, respectively). Histograms also represent inhibition by ω-agatoxin of Ca 2+ transients for WT ( n = 14) and KI ( n = 35) neurons. After ω-agatoxin responses of WT and KI neurons differ from their own controls (** p ≤ 0.001). C , Representative traces of α,β-meATP (10 μ M, 2-s application)-evoked Ca 2+ transients before ( black trace ) and after ( red trace ) application of ω-agatoxin (200 nM, 30 min). D , Histograms show larger Ca 2+ transients evoked by α,β-meATP (10 μ M, 2-s application) from KI ( n = 26) than WT ( n = 16) and neurons (* p = 0.04). Histograms also show that ω-agatoxin reduced Ca 2+ transients of KI ( n = 22) and WT ( n = 9) neurons. ** p ≤ 0.001 for each case. E , Microphotographs of immunofluorescence experiments depicting WT and KI trigeminal neurons in culture expressing P2X 3 receptors or Ca V 2.1 channels. Bar = 50 μ m. Histograms ( right ) show% of P2X 3 - (top) or Ca V 2.1- (bottom) immunoreactive neurons (taking as 100% the β-tubulin III immunoreactive) ( n = 5, p > 0.05 for P2X 3 receptors; n = 3, p > 0.05 for Ca V 2.1-expressing neurons). F , Histograms show% of Ca V 2.1-immunoreactive neurons ( top ; taken as 100%) which are immunopositive for P2X 3 ( n = 7, p > 0.05) or% of P2X 3 -immunoreactive neurons ( bottom ) which are immunopositive for Ca V 2.1 (n = 4, p > 0.05).

Article Snippet: For immunofluorescence microscopy, the following antibodies were used: anti-P2X 3 , anti-P2X 2 , anti-TRPV1 and anti-Ca V 2.1 α1 (1:200; all from Alomone Labs, Jerusalem, Israel); anti-P2X 3 (1:300; Neuromics, Edina, MN, USA), anti-β-tubulin III (1:1000; Sigma), anti-phosphorylated Thr286 CaMKII (1:500; Promega, Madison, WI, USA), anti-phosphorylated Ser133 CREB (1:300; Upstate Millipore, NY, USA).

Techniques: Inhibition, Immunofluorescence, Expressing

Journal: Molecular Pain

Article Title: Familial hemiplegic migraine Ca V 2.1 channel mutation R192Q enhances ATP-gated P2X 3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

doi: 10.1186/1744-8069-6-48

Figure Lengend Snippet: Survival of WT and KI trigeminal neurons in culture and their expression of Ca v 2.1 protein . A , Survival is calculated as number of β-tubulin III positive cells per unit area after 1-4 days in culture. Data are normalized with respect to those at 1 day. n = 4, p > 0.05. B , Somatic size distribution of trigeminal neurons (β-tubulin III immunoreactive) in culture from WT and KI mice. n = 4. C , Immunocytochemical expression of Ca V 2.1 channels in intact trigeminal ganglia of WT and KI mice. Histograms represent% of Ca V 2.1 immunoreactive neurons over β-tubulinIII immunoreactive neurons in WT or KI ganglia. n = 3, p > 0.05. D , Example of western blot of protein extracts from WT and KI trigeminal ganglia or culture, probed with anti-Ca V 2.1 antibody. Equal loading was ensured by membrane probing with β-tubulinIII antibodies. n = 3, p > 0.05. Histograms ( right ) show no significant difference between these conditions.

Article Snippet: For immunofluorescence microscopy, the following antibodies were used: anti-P2X 3 , anti-P2X 2 , anti-TRPV1 and anti-Ca V 2.1 α1 (1:200; all from Alomone Labs, Jerusalem, Israel); anti-P2X 3 (1:300; Neuromics, Edina, MN, USA), anti-β-tubulin III (1:1000; Sigma), anti-phosphorylated Thr286 CaMKII (1:500; Promega, Madison, WI, USA), anti-phosphorylated Ser133 CREB (1:300; Upstate Millipore, NY, USA).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

doi: 10.1101/2022.07.22.501145

Figure Lengend Snippet: ( A-B ) Specificity of PI(4,5)P 2 labeling. Replicas of liposomes containing 5% PtdIns or different stereoisomers of PIs were labeled using GST-PH, anti-GST antibody, and 5-nm gold particle-conjugated secondary antibody ( A , Scale bar = 200 nm). The density of gold particles was highest in the liposome containing PI(4,5)P 2 ( B ). (C) Acute cerebellar slice preparation for high-pressure freezing (HPF) and replica preparation. Left-top, an acute sagittal slice of the mouse cerebellum. The dashed line indicates the trimmed region for HPF. Left-bottom, a trimmed cerebellar slice on a copper carrier with double-sided tape for HPF. Right, low-magnification transmission electron microscopic (TEM) image of the mouse cerebellar replica containing granule cell layer (GCL), Purkinje cell layer (PCL), and molecular layer (ML). Scale bar = 20 µm. (D) Example TEM image of 5-nm gold particle labeling for PI(4,5)P 2 with immunogold labeling for Ca V 2.1 (12 nm) on P- (left) and E-face (right) of the PC somatic membranes of cerebellar PC. Scale bar = 200 nm. (E) Statistical comparison of the PI(4,5)P 2 particle density on the E-face and P-face of the PC somatic membranes. Open and closed circles indicate the means of the PI(4,5)P 2 particle density in each PC and each mouse, respectively, with different colors. Black horizontal bars indicate estimated marginal means (emmeans, thick bars) and 95% confidence intervals (CIs, error bars) of the density estimated by GLMM (Methods). The PI(4,5)P 2 density was significantly higher on the P-face than on the E-face of the PC somatic membranes (P-face: 51.2 ± 8.5 particles/µm 2 , E-face: 8.0 ± 1.2 particles/µm 2 , n = 213 images/12 cells/4 mice, p < 0.001, Chi-square likelihood ratio test (Chi-LRT)). (F) Comparison of nearest neighbor distances (NND) between real (Data NND, x-axis) and simulated (Sim NND, y-axis) PI(4,5)P 2 particles on PC somatic membranes. Data-NNDs are significantly smaller than Sim NNDs (Data: 35.1 ± 3.6 nm, Sim: 73.3 ± 7.0 nm, n = 206 images/11 cells/4 mice, p < 0.001, Chi-LRT). (G) Distribution of NNDs of the PI(4,5)P 2 particles obtained from a single PC somatic membrane (n = 2,929 particles). Red and blue lines indicate the distinct components of the NND distribution estimated from the Gaussian mixture modeling. (H) PI(4,5)P 2 labeling (5 nm) with Ca V 2.1 (12 nm) on the P-face of GC (left) and molecular layer interneuron (MLI, right) somatic membranes. Scale bar = 200 nm. (I) The PI(4,5)P 2 particle density on the somatic membranes (P-face) of different neuronal cell types in the mouse cerebellum (PC: 51.8 ± 9.6 particles/µm 2 , GC: 69.8 ± 11.3 particles/µm 2 , MLI: 42.9 ± 6.7 particles/µm 2 , n = 273 images/57 cells/4 mice, p = 0.003, Chi-LRT). (J) NND of the PI(4,5)P 2 particles on the somatic membranes of different neuronal cell types in the mouse cerebellum (PC: 34.4 ± 3.0 nm, GC: 31.0 ± 2.4 nm, MLI: 34.8 ± 2.6 nm, n = 65,607 values/57 cells/4 mice, p = 0.12, Chi-LRT). Figure Supplement 1 . Non-specific labeling on the somatic membranes of PCs

Article Snippet: Then the replica was incubated with anti-GST antibody (rabbit IgG, 5 µg/ml, Bethyl Laboratories) and anti-Ca V 2.1 antibody (guinea pig, 2.5 µg/ml, Synaptic Systems) as a marker of neurons at 15°C overnight, and then gold-nanoparticle conjugated secondary antibodies (goat anti-rabbit IgG, 5 nm, 1:50, BBI; donkey anti-guinea pig IgG, 12 nm, 1:30, Jackson Immnoresearch) dissolved in the dilution buffer at 15°C overnight.

Techniques: Labeling, Slice Preparation, Transmission Assay

Journal: bioRxiv

Article Title: Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

doi: 10.1101/2022.07.22.501145

Figure Lengend Snippet: (A) Example images for PI(4,5)P 2 particle distribution on the membranes of PF-PC (top) and PF-MLI bouton (bottom). Red and black circles indicate gold particles for PI(4,5)P 2 and Ca V 2.1, respectively. The Blue area and dotted line indicate AZ and outer-rim (30 nm from the edge of AZ, Methods), respectively. Scale bar = 200 nm. (B) Beeswarm plot of the PI(4,5)P 2 density in the whole bouton (Bt), AZs, and extra-AZ region (exAZ) of the PF-PC (left) and PF-MLI (right) bouton membranes. The PI(4,5)P 2 density was significantly higher in AZs than in the whole bouton and the exAZ in both PF-PC and PF-MLI boutons (n = 111 boutons/4 mice, p < 0.001, Tukey method). (C) Comparison of the PI(4,5)P 2 density in the AZs between real and simulated random distribution on the PF bouton membranes. The density of the real particle distribution was significantly higher than that of the simulated one in both PF-PC (real: 97.2 ± 19.7 particles/µm 2 , sim: 66.7 ± 13.5 particles/µm 2 , n = 55 boutons/4 mice, p < 0.001, Chi-LRT) and PF-MLI (real 86.7 ± 12.4 particles/µm 2 , sim: 73.2 ± 10.4 particles/µm 2 , n = 56 boutons/4 mice, p = 0.002, Chi-LRT) AZ membranes. (D) Distribution of center-periphery index (CPI) of PI(4,5)P 2 particles in boutons (top) and AZs (bottom). Blue in the bottom graph indicates the CPI distribution of the simulated particles that are randomly distributed in AZs. (E) Comparison of CPIs of the PI(4,5)P 2 particles in AZs between PF-PC and PF-MLI AZs. There is no significant difference in the CPIs between PF-PC and PF-MLI AZs (PF-PC: 0.67 ± 0.02, PF-MLI: 0.67 ± 0.02, n = 107 boutons/4 mice, p = 0.999, Chi-LRT). Dashed lines with red show the mean CPIs of the simulated randomly-distributed particles. Asterisks on the bars indicate statistical differences in CPIs between real and simulated particles (PF-PC: 0.59 ± 0.02, PF-MLI: 0.58 ± 0.02, *p < 0.05, **p < 0.01, Tukey method). (F) Example images of the PI(4,5)P 2 particle distribution on the PC spine membrane. Red and black circles indicate PI(4,5)P 2 and GluD2, respectively. The green area indicates postsynaptic density (PSD) based on the cluster of GluD2. Scale bar = 200 nm. (G) Beeswarm plot of the PI(4,5)P 2 density in the whole spine (spine), PSD, and extra-PSD region (exPSD) of the PC spine membranes. No significant difference in the density was detected between these compartments (n = 108 spines/6 mice, p = 0.77, Chi-LRT). (H) Comparison of the PI(4,5)P 2 density in the PSDs between real and simulated random distribution on the PC spine membranes. The density of the real and simulated particle distribution was not significantly different (real: 0.59 ± 0.01, sim: 0.55 ± 0.01, n = 108 spines/6 mice, p = 0.25, Chi-LRT). (I) Distribution of CPIs of PI(4,5)P 2 particles in spines (top) and PSDs (bottom). Blue in the bottom graph indicates the CPI distribution of the simulated particles that are randomly distributed in PSDs. The CPI of the PI(4,5)P 2 particles is uniformly distributed in the spines and PSDs, suggesting the random distribution of PI(4,5)P 2 . Table Supplement 1 . PI(4,5)P 2 particle density in synaptic membranes of cerebellar neurons

Article Snippet: Then the replica was incubated with anti-GST antibody (rabbit IgG, 5 µg/ml, Bethyl Laboratories) and anti-Ca V 2.1 antibody (guinea pig, 2.5 µg/ml, Synaptic Systems) as a marker of neurons at 15°C overnight, and then gold-nanoparticle conjugated secondary antibodies (goat anti-rabbit IgG, 5 nm, 1:50, BBI; donkey anti-guinea pig IgG, 12 nm, 1:30, Jackson Immnoresearch) dissolved in the dilution buffer at 15°C overnight.

Techniques:

Journal: bioRxiv

Article Title: Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

doi: 10.1101/2022.07.22.501145

Figure Lengend Snippet: (A) Example images for co-immunolabeling of PI(4,5)P 2 and Ca V 2.1 on somatic (left), SpB (middle), and spine (right) membranes of the PC. Red and purple (closed, open) circles indicate PI(4,5)P 2 and Ca V 2.1 (real, fitted-simulated) particles, respectively. Scale bars = 200 nm. (B) Comparison of the NNDs from Ca V 2.1 to PI(4,5)P 2 particles (NND C-P ) between real and fitted-simulated Ca V 2.1 distribution on somatic (left), SpB (middle), and spine (right) membranes of PCs. The NND C-P of the real distribution was significantly smaller than that of the simulated one in somatic (real: 89.7 ± 9.6 nm, sim: 112.9 ± 12.1 nm, n = 129 images/14 cells/5 mice, p < 0.001, Chi-LRT), SpB (real: 82.3 ± 8.1 nm, sim: 100.5 ± 9.9 nm, n = 158 images/20 dendrites/4 mice, p < 0.001, Chi-LRT), and spine membrane (real: 80.9 ± 8.7 nm, sim: 92.8 ± 9.9 nm, 69 spines/4 mice, p = 0.006, Chi-LRT). (C) Comparison of NND C-P between somatodendritic compartments. There is no significant difference between the PC compartments (n = 12,956 values/92 components/5 mice, p = 0.70, Chi-LRT). (D) Example images for co-immunolabeling of PI(4,5)P 2 and Ca V 2.1 on somatic (left) and presynaptic PF-PC (middle) and PF-MLI (right) AZ membranes of the GC. Red and purple (closed, open) circles indicate PI(4,5)P 2 and Ca V 2.1 (real, fitted-simulated) particles, respectively. The Blue area and dotted lines indicate AZs and the outer-rim, respectively. Scale bars = 200 nm. (E) Comparison of the NND C-P between real and fitted-simulated Ca V 2.1 distribution on somatic (left), PF-PC AZ (middle), and PF-MLI AZ (right) membranes of GCs.The NND C-P of the real distribution was significantly smaller than that of the simulated one in somatic (real: 76.0 ± 9.0 nm, sim: 93.0 ± 13.1 nm, n = 81 images/25 cells/5 mice, p = 0.017, Chi-LRT), PF-PC AZ (real: 54.0 ± 7.9 nm, sim: 64.0 ± 9.3 nm, n = 54 AZs/4 mice, p < 0.001, Chi-LRT), and PF-MLI AZ membrane (real: 60.8 ± 6.7 nm, sim: 67.0 ± 7.3 nm, 52 AZs/4 mice, p = 0.006, Chi-LRT). (F) Comparison of NND C-P between presynaptic AZs of PF-PC and PF-MLI synapses. There is no significant difference in NND C-P between the AZs (n = 1,110 values/68 AZs/4 mice, p = 0.60, Chi-LRT).

Article Snippet: Then the replica was incubated with anti-GST antibody (rabbit IgG, 5 µg/ml, Bethyl Laboratories) and anti-Ca V 2.1 antibody (guinea pig, 2.5 µg/ml, Synaptic Systems) as a marker of neurons at 15°C overnight, and then gold-nanoparticle conjugated secondary antibodies (goat anti-rabbit IgG, 5 nm, 1:50, BBI; donkey anti-guinea pig IgG, 12 nm, 1:30, Jackson Immnoresearch) dissolved in the dilution buffer at 15°C overnight.

Techniques: Immunolabeling

Journal: bioRxiv

Article Title: Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

doi: 10.1101/2022.07.22.501145

Figure Lengend Snippet: (A) Example images for co-immunolabeling of PI(4,5)P 2 and Ca V 2.1 on somatic (left) and basket cell (BC)-PC bouton (right) membranes of the MLI. Red and purple (closed, open) circles indicate PI(4,5)P 2 and Ca V 2.1 (real, fitted-simulated) particles, respectively. P-face of BC-PC boutons were identified based on Ca V 2.1 clusters and the surrounding E-face of the PC somatic membranes with Ca V 2.1 clusters. Scale bars = 200 nm. (B) Comparison of the NND C-P between real and fitted-simulated Ca V 2.1 distribution on somatic (left) and BC-PC bouton (right) membranes of MLIs. The NND C-P of the real distribution was significantly smaller than that of the simulated one in somatic (real: 97.5 ± 13.1 nm, sim: 115.4 ± 15.5 nm, n = 102 images/26 cells/4 mice, p < 0.001, Chi-LRT) and BC-PC bouton membrane (real: 106.0 ± 7.7 nm, sim: 143.0 ± 10.4 nm, 37 boutons/3 mice, p = 0.006, Chi-LRT). (C) Comparison of NND C-P between somatic and bouton membranes. No significant difference was shown between these compartments (n = 2,854 values/63 components/4 mice, p = 0.29, Chi-LRT).

Article Snippet: Then the replica was incubated with anti-GST antibody (rabbit IgG, 5 µg/ml, Bethyl Laboratories) and anti-Ca V 2.1 antibody (guinea pig, 2.5 µg/ml, Synaptic Systems) as a marker of neurons at 15°C overnight, and then gold-nanoparticle conjugated secondary antibodies (goat anti-rabbit IgG, 5 nm, 1:50, BBI; donkey anti-guinea pig IgG, 12 nm, 1:30, Jackson Immnoresearch) dissolved in the dilution buffer at 15°C overnight.

Techniques: Immunolabeling

Journal: bioRxiv

Article Title: Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

doi: 10.1101/2022.07.22.501145

Figure Lengend Snippet: (A) Example images of the PI(4,5)P 2 labeling without incubation with GST-PH on the P- (left) and E-face of PC somatic membranes. The replicas were subsequently incubated with anti-GST/anti-Ca V 2.1 primary antibodies and gold-conjugated secondary antibodies (5 nm for anti-GST, 12 nm for anti-Ca V 2.1). Scale bar = 200 nm. (B) Comparison of the 5-nm gold particle densities with and without GST-PH on the E- and P-face of PC somatic membranes. The particle density without GST-PH was significantly lower than that with GST-PH in both faces. Without the GST-PH incubation, no significant difference in the density was detected between the E- and P-face (p = 0.86, multiple comparisons with the Benjamini-Hochberg (BH) method). (C) Comparison of the number of 5-nm gold particles per cluster on the E- and P-face of the PC somatic membranes incubated with or without GST-PH. The clusters were detected using Ward Linkage hierarchical clustering method. The threshold distance to separate each cluster was set as 50 nm, which is nearby the maximum distance for cluster detection on the PC somatic membrane using DBSCAN ( , Method). The particle number per cluster without GST-PH incubation was significantly lower than that with GST-PH in both faces (multiple comparisons with BH method), suggesting that the PI(4,5)P 2 particle clusters observed on the neuronal membranes are also ascribable to specific clustering of GST-PH binding sites but not to non-specific aggregation of the primary and secondary antibodies.

Article Snippet: Then the replica was incubated with anti-GST antibody (rabbit IgG, 5 µg/ml, Bethyl Laboratories) and anti-Ca V 2.1 antibody (guinea pig, 2.5 µg/ml, Synaptic Systems) as a marker of neurons at 15°C overnight, and then gold-nanoparticle conjugated secondary antibodies (goat anti-rabbit IgG, 5 nm, 1:50, BBI; donkey anti-guinea pig IgG, 12 nm, 1:30, Jackson Immnoresearch) dissolved in the dilution buffer at 15°C overnight.

Techniques: Labeling, Incubation, Binding Assay

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: a Representative confocal image of a nerve terminal arborization. Singly, dually, and innervated by three or more axons NMJs from YFP muscles and also images of the morphologic maturation (S1, the most inmature, and S4, almost fully differentiated, stages) of the postsynaptic clusters from P9 mice. The bar indicates 10 μm. b Confocal immunofluorescence location of α 1D L-, N-, and P/Q-type voltage-dependent calcium channels (VDCCs) at the NMJ. Triple labeling of VDCCs (green fluorescence) with syntaxin (blue fluorescence) and nAChR-α-bungarotoxin (red fluorescence) in merge images. Figure shows the presence of α 1D L-, N-, and P/Q-type-VDCC (in green) in the nerve terminal of P9 Levator auris longus (LAL) muscle endplates. The bar indicates 10 μm

Article Snippet: Muscles were incubated overnight at 4 °C with anti-Ca V 1.3 (CACNA1D) antibody voltage-dependent L-type calcium channel subunit α 1D (1/100; ACC-005, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.1 (CACNA1A) antibody voltage-dependent P/Q-type calcium channel subunit α 1A (1/100; ACC-001, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.2 (CACNA1B) antibody voltage-dependent N-type calcium channel subunit α 1B (1/100; ACC1-002, Alomone Labs, Jerusalem, Israel), and anti-mouse syntaxin (1/1000, S066, Sigma, St Louis, MO, USA).

Techniques: Immunofluorescence, Labeling, Fluorescence

Journal: Frontiers in Neuroanatomy

Article Title: Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images

doi: 10.3389/fnana.2022.855218

Figure Lengend Snippet: Application of Gold Rippler and Gold Star for synaptic protein analysis. (A) Ca V 2.1 and Munc13-1 colocalize at intramembrane protein-rich regions of the calyx of Held membrane which presumably represent the presynaptic active zone. Only a small portion of the larger membrane face (ROI) is shown. (B) Overall gold particle density across the calyx of Held membrane for two different SDS-digestion conditions. Gray data points represent individual ROIs and gray lines connect Ca V 2.1 and Munc13-1 densities coming from the same ROI. Solid black dots represent mean densities. Ca V 2.1 and Munc13-1 densities from the ROI partially shown in (A) are indicated by light blue circles. (C) Zoomed view of the gray box in (A) showing a conceptual illustration of Gold Rippler for visual clarity. (D) LCPI curve for real and random particle distributions and two SDS-digestion conditions, where Munc13-1 particles are set as landmarks. (E) Zoomed view of the gray box in (A) showing an illustration of Gold Star. When Munc13-1 is set as the landmark, NNDs (blue lines) are measured between each Ca V 2.1 and the closest Munc13-1 particle (blue dots). (F) Cumulative frequency distribution of real and random particle-particle NNDs pooled across the 6 ROIs for each SDS-digestion condition. (G) Grand mean particle-particle NND for each SDS-digestion condition. Error bars represent S.E.M. n = 6 ROIs for each condition. Scale bars: 100 nm.

Article Snippet: The replicas were then incubated at room temperature for overnight with following antibodies per each experiment: (1) cerebellum replicas—a guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody (Synaptic Systems, Göttingen, Germany; 152–205 at 0.7 μg/ml), (2) brain stem replicas used for the large terminal image analysis—a guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody (Synaptic Systems, Göttingen, Germany; 152–205 at 0.7 μg/ml) and rabbit anti GFP antibody (Abcam, Cambridge, United Kingdom; ab6556 at 1μg/ml), 3) brain stem replicas used for Ca V 2.1 and Munc13-1 localization analysis—the same guinea pig anti Ca V 2.1+ channel P/Q-type alpha-1A antibody and a rabbit anti Munc13-1 antibody (Synaptic Systems, Göttingen, Germany; 126–103 at 1 μg/ml).

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