Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Immunocytochemical analysis of the spatial relationships between peripheral cytoplasmic Y172 immunolabeling and excitatory and inhibitory afferent inputs to MNs of CD1 mice. Spinal cord sections were double immunostained with Y172 (green) and either anti-VAChT, VGluT1 or VGAT (for cholinergic, glutamatergic or GABAergic synapses, respectively, all red) and processed for fluorescent Nissl staining for MN visualization (blue). (A1–A5) Representative maximum intensity projections from confocal Z-stacked images showing Y172 and VAChT immunoreactivity in an MN of an adult (P75) mouse. Note the distribution of Y172-positive spots in the cytoplasm of the cell body; while some immunoreactive patches were located around the nucleus, others were peripherally distributed and exhibited a close association with VAChT-positive puncta. The area delimited by the dotted-lined rectangle in (A2) is shown at higher magnification in (A3–A5) ; note that, while the peripherally located Y172-positive spot was in contact with a VAChT-positive punctum, the spot that was more internally located did not exhibit any association with VAChT immunolabeling. (B,C) Pixel profile analysis (C) along a line crossing a multifluorescent-labeled VAChT/Y172 synapse (shown in B ) demonstrating the dissociation of presynaptic VAChT immunostaining and postsynaptic Y172-positive staining; the blue channel, corresponding to fluorescent Nissl staining, is not included in the graph. (D) Volume rendering of a high magnification confocal image of a C-bouton double immunolabeled with anti-VAChT (red) and Y172 antibodies (green) demonstrating the nonoverlapping and separate distribution of both signals; the blue channel corresponds to fluorescent Nissl stain for MN visualization. (E1–F5) Representative Z-staked images showing MNs immunostained with Y172 and either anti-VGluT1 (E1–E5) or anti-VGAT (F1–F5) antibodies. Note that neither VGluT1- nor VGAT-containing puncta were associated with Y172-positive profiles; the occasional degree of pixel overlapping observed in some cases was due to the random close proximity between Y172 and VGluT1 or VGAT immunoreactivity. (G–J) Pixel profile analysis (H,J) along the lines depicted in (G,I) ; in (G) , the yellow line delimits the periphery of an MN by passing through different Y172- and VGluT1-positive spots, whereas in (I) , the lines cross two spots with VGluT1 immunoreactivity (1, red) and one spot with Y172 immunoreactivity (2, green); note the absence of colocalization between the two signals in (H,J) . (K) The time course of changes in the number of Y172- and VAChT-positive profiles per 100 μm of soma perimeter in spinal cord MNs from mice at different ages. (L–N) The percentage of peripheral Y172-positive profiles closely associated with puncta positive for VAChT, VGluT1, or VGAT, and vice versa, in adult (P75) MNs is shown in (L–N) , respectively. The data are expressed as the mean ± SEM; 200–300 profiles from 10 to 15 randomly selected MNs (3–4 animals) per condition were analyzed; * p < 0.05 vs. Y172+ immunoreactivity; student’s t -test. Scale bars: A1 = 10 μm (valid for A2 ); D = 1 μm; E1,F1 = 10 μm (valid for E2,F2 ); F5 = 1.5 μm (valid for A3–A5,E3–E5,F3,F4 ); G = 10 μm.

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Immunolabeling, Staining, Labeling, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Changes in Y172 immunoreactivity in axotomized MNs. The sections of the spinal cord from adult (P60) mice subjected to unilateral sciatic nerve transection were immunostained with the Y172 antibody in combination with antibodies against VAChT (blue) and IBA1 (red), as indicated in the panels, to identify C-boutons and microglia, respectively; sections were also counterstained with fluorescent Nissl stain for MN visualization (shown in gray in F4,G4 ). (A1–B2) Ventral horn MNs in general view (A1–B2 ) and higher magnification (C1–E5) images from the contralateral (contra., nonaxotomized, A1, A2,C1–C5 ) and ipsilateral (ipsi., axotomized, B1, B2,D1–E5 ) sides of the spinal cord 1 (A1–D5) and 14 (E1–E5) days after nerve transection; note the nuclear Y172 immunostaining (B1,D1,E1) and the prominent IBA1-positive reactive microglia on the ipsilateral side (B2, D4,E4) in relation to those on the contralateral side (A1,A2,C1,C4) ; in (E1) , an MN with intense nuclear Y172 staining as well as numerous profiles immunoreactive with the monoclonal antibody can be seen. The areas delimited by dotted-line rectangles in (C4,D4,E4) are shown at higher magnifications in (C5,D5,E5) ; note, on the ipsilateral side (D4,E4) , the presence of abundant IBA1-positive microglial processes enwrapping MNs and contacting Y172-positive profiles, suggesting a role for microglia in the loss of peripheral cytoplasmic Y172 immunolabeling in axotomized MNs. (F1–G4) MNs on the ipsilateral side of spinal cord 30 (F1–F4) and 180 (G1–G4) days after axotomy; note in (F1–F4) the intense nuclear signal and the depletion of cytoplasmic peripheral profiles positive with the Y172 antibody. In (G1–G4) , an apparently healthy MN exhibiting both abundant cytoplasmic profiles and the absence of nuclear immunostaining with the Y172 antibody can be seen in the vicinity of an atrophic MN showing intense nuclear Y172 immunoreactivity. In all cases, MNs with nuclear Y172 signal displayed a reduced density of VAChT-positive C-boutons. (H,I ) The density (per 100 μm 2 of cell body) and size (in μm 2 ) of cytoplasmic Y172-positive profiles in MNs located on the contralateral and ipsilateral sides of the spinal cord on different days after unilateral sciatic nerve transection. (J) Changes (expressed as the % of the contralateral side) in the density of VAChT-positive puncta observed in ipsilateral side MNs on different days post-axotomy. The data in the graph are expressed as the mean ± SEM, * p < 0.05 and *** p < 0.001 vs. the contralateral side; n = 8–11 randomly selected MNs per side from three mice per day post-axotomy. Scale bars: B2 = 40 μm (valid for A1–B1 ); E4 = 20 μm (valid for C1–C4,D1–D4,E1–E3 ); E5 = 5 μm (valid for C5,D5,E5 ); G4 =10 μm (valid for F1–G3 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Staining, Immunostaining, Immunolabeling

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Changes in Y172 immunoreactivity in MNs from mutant mice (P60) overexpressing NRG1 type III. (A–F) The density (per 100 μm 2 MN soma, A,B ) and size (in μm 2 , C,D ) of total (A,C) and peripheral (periph., B,D ) Y172-positive profiles and the percentage of these profiles showing a spatial association with VAChT-positive C-boutons (E) and NRG1 type III-positive spots (F) in MNs from WT and NRG1 type III-overexpressing mice. Note that NRG1 type III overexpression was associated with a prominent decrease in the density of total Y172-positive profiles (A) and a significant increase in the number of those located peripherally (B) in MNs; the area of both total and peripheral Y172-positive profiles was dramatically increased in MNs from NRG1 type III-overexpressing animals (C,D) . Additionally, the percentage of Y172-positive profiles showing a close association with VAChT- (E) or NRG1 type III-positive (F) spots significantly increased or decreased, respectively, in MNs from NRG1 type III-overexpressing animals; 10–15 randomly selected MNs from 3 to 4 mice per condition were analyzed; * p < 0.05 and *** p < 0.001 vs. WT; student’s t -test). ( G1–G4) Representative confocal micrographs of an NRG1 type III-overexpressing MN immunostained with Y172 (green) and anti-NRG1 type III (red) antibodies and counterstained with fluorescent Nissl stain (blue) for neuron visualization. Note that, compared to MNs of adult CD1 mice (see, for instance, or ), MNs overexpressing NRG1 type III exhibit an enlargement of Y172-positive profiles located in the periphery of the cell body, which correlated with the redundant and expanded SSCs previously described in Salvany et al. ; note also the expansion of NRG1 type III immunolabeling peripherally located in MN soma. (H1–H3) A higher magnification image of the area delimited in (G4) by the dotted-line rectangle corresponding to Y172, NRG1 type III and merged channels, as indicated, is shown. Scale bars: G4 = 10 μm (valid for G1-G3 ); H3 = 2.5 μm (valid for H1, H2 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Mutagenesis, Over Expression, Staining, Immunolabeling

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Changes in Y172 immunoreactivity in MNs of SOD1 G93A mice. (A–C) The density (number per 100 μm of soma perimeter) of peripheral Y172-positive profiles (shown as the % of WT, A ) and of VAChT-positive puncta (B) , and the percentage of VAChT-positive puncta closely associated with Y172-positive profiles (C) in WT and mutant MNs at different stages of disease (presymptomatic [P60], early symptomatic [P90], and end-stage [P120]). The data are expressed as the mean ± SEM; 15–22 MNs randomly selected from SOD1 G93A animals and respective WT littermates were analyzed; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. WT, student’s t -test. (D1–E4) Representative confocal micrographs of spinal cord sections from SOD1 G93A mice at the presymptomatic (P60, D1–D4 ) and end (P120, E1–E4 ) stages of disease immunostained with Y172 (green), anti-IBA1 (for microglia, red) and anti-VAChT (for C-boutons, blue) antibodies, as indicated in the panels; sections were also counterstained with fluorescent Nissl stain (blue in D4 ) for MN visualization. Note the presence of two MNs (delimited with dotted lines in D3 ), one of them exhibiting a normal appearance with abundant Y172-positive profiles widely distributed in the cell body cytoplasm and another with a typical appearance displaying intense nuclear labeling with the Y172 antibody and almost a complete absence of cytoplasmic profiles immunostained with this antibody, in (D1–D4) ; there were numerous IBA1-positive microglial processes around the second type of MN, suggesting incipient degenerative changes. Prominent reactive microgliosis found around degenerating MNs exhibiting nuclear Y172 immunostaining in a P120 SOD1 G93A mouse is shown in (E1–E4) . (F1–F4) A spinal cord MN from a SOD1 G93A mouse at end-stage (P120) showing strong nuclear Y172 immunoreactivity (green) but depletion of cytoplasmic profiles immunostained with the Y172 antibody; this MN, however, still exhibited abundant VAChT-positive C-boutons (red), with the vast majority of them not displaying any association with the scarce Y172-positive profiles remaining. VAChT-positive C-boutons delimited with dotted-line squares in ( F3 ; indicated as 1 and 2) are shown in (G1–G3) (1) and (H1–H3) (2); C-bouton number 1 was associated with a small remnant Y172-positive signal, whereas, Y172-immunoreactivity was completely lost in C-bouton number 2. Scale bars: D4,E4 = 20 μm (valid for D1–D3,E1–E3 , respectively); F4 = 10 μm (valid for F1–F3 ); H3 = 1 μm (valid for G1–H2 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Mutagenesis, Staining, Labeling, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Changes in Y172 immunoreactivity in MNs from Smn 2B/- mice. (A–C) The density (per 100 μm of cell body perimeter) of peripherally located Y172-positive profiles (A) and of VAChT-positive puncta (B) , and the percentage of VAChT-positive puncta spatially associated with Y172 immunoreactivity (C) in mutant mouse MNs at different stages of disease (early presymptomatic [P1 and P5], late symptomatic [P20], and end-stage [P25–30]). (D1–E4) Representative confocal micrographs of spinal cord sections from Smn 2B/- mice at P25 immunostained with Y172 (green) and anti-VAChT (for C-boutons, red) antibodies, as indicated in the panels; sections were also counterstained with fluorescent Nissl stain (blue in D4 and E4 ) for MN visualization. Note the intense nuclear Y172 immunostaining in (D1,E1) and the marked depletion in Y172-positive profiles found in spinal muscular atrophy (SMA) MNs.The data are expressed as the mean ± SEM; 15–20 MNs randomly selected from Smn 2B/- mice and respective WT littermates were analyzed; ** p < 0.01 and *** p < 0.001 vs. WT, student’s t -test.Scale bars: D4 = 40 μm (valid for D1–D3 ); and E4 = 10 μm (valid for E1–E3 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Mutagenesis, Staining, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: p-c-Jun-like immunodetection with antibodies other than Y172 under basal conditions and in axotomized MNs (30 days after sciatic nerve transection at P60). (A1–E4) Representative images of spinal cord MNs double immunostained with different polyclonal antibodies (pAbs) against c-Jun (green) phosphorylated at either Ser63 (A1–B4) or Ser73 (C1–E4) and VAChT (red); fluorescent Nissl stain (blue in A4,B4,C4,D4 , and E4 ) was used to visualize MNs. The images in (A1,B1) , which show the p-c-Jun (Ser63) channel, were obtained following the modification of scanning parameters to achieve a higher sensitivity of detection than that used for the Y172 antibody. Note the absence of p-c-Jun (Ser63) nuclear immunostaining and the presence of immunolabeled cytoplasmic profiles mainly located in the periphery of the cell body (arrows in A3,A4 ) under basal conditions (A1–A4) . Thirty days after unilateral sciatic nerve transection (B1–B4) , the same antibody revealed prominent nuclear immunostaining in MNs located on the ipsilateral (operated) side of the spinal cord (white arrow in B4 ) but an almost complete absence of cytoplasmic positive profiles. The intense immunoreactivity in the nucleus of the presumably axotomized MN was in contrast with the faint nuclear immunostaining in the neighboring neuron, likely corresponding to a nonlesioned MN (blue arrow in B4). (C1–C4) Negative immunostaining with the polyclonal antibody against p-c-Jun (Ser73) was observed in both the nucleus and cytoplasm of an MN under basal conditions. (D1–E4) The same antibody showed intense positive nuclear immunostaining and the absence of immunostained cytoplasmic profiles in axotomized MNs, both in the ipsilateral and contralateral side of the spinal cord. (F) Representative western blots of mouse spinal cord extracts probed with the antibodies against p-c-Jun used in our study; a monoclonal antibody against c-Jun (c-Jun mAb) was also included, and β-actin was used as a loading control. Note that a band corresponding to ~43–48 kDa, as expected for p-c-Jun, was observed; the same band was also seen in western blots performed with the anti-c-Jun antibody. (G) Densitometric analysis of p-c-Jun bands obtained in western blots by probing with the antibodies used for analysis. The data were normalized to β-actin; the bars represent the values (mean ± SEM) of extracts from three mice. Scale bar: E4 = 20 μm (valid for A1–E3 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Immunodetection, Staining, Modification, Immunostaining, Immunolabeling, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons

doi: 10.3389/fncel.2019.00582

Figure Lengend Snippet: Western blot and immunocytochemical analyses with the Y172 antibody in NSC-34 cells (A–E4) and cultured MNs (F–K4) . (A) The time course of Y172 antigen expression levels in NSC-34 cells after a different number of days (0, 2, 4 and 8) (d) of differentiation, as determined by western blot analysis; the densitometric analysis was normalized to β-actin, which was used as a loading control. (B) Representative western blots of NSC-34 cells probed with the Y172 antibody used for quantification in (A) . (C1–E4) Representative confocal images of NSC-34 cells after 2 (C1–C4) , 4 (D1–D4) and 8 (E1–E4) days of differentiation stained with Y172 (green) and anti-VAChT (red) antibodies as well as DAPI (blue) for DNA, as indicated. Y172 immunostaining was found exclusively in the nuclei of NSC-34 cells following 2 days of differentiation (C1–4) ; however, granular immunostaining was observed in the cytoplasm of cell bodies and neurites after 4 days of differentiation (D1–4) ; after 8 days of differentiation, Y172 immunostaining decreased in the nucleus but markedly increased in the cytoplasm of cell bodies and proximal neurites (E1–4) . VAChT immunostaining displayed a diffuse and faintly positive pattern into the cell body cytoplasm, and although some positive puncta appear to be present, these were very scarce and did not have a defined location in NSC-34 cells. (F) The time course of Y172 antigen expression levels in MNs cultured for 3, 6 and 12 days, determined by western blot analysis; the densitometric analysis was normalized to β-actin, which was used as a loading control. (G) Representative western blots of cultured MNs probed with the Y172 antibody used for quantification in (F) . ( H1–K4) Representative confocal images of MNs cultured for 3 (H1–4) , 6 (I1–4) and 12 (J1–4) days stained with Y172 (green) and anti-VAChT (red) antibodies as well as DAPI (blue) for DNA, as indicated. Note the intense nuclear positivity but the faint and discrete cytoplasmic signal obtained with the Y172 antibody at 3 DIV (H1–4) ; however, at 6 DIV (I1–I4) , an increase in the cytoplasmic immunolabeling, mainly in the form of oval patches in the soma and proximal neurites, was observed. Following 12 days of culture (J1–4) , a reduction in Y172 immunolabeling was observed in the cytoplasm, although some positive profiles remained. The area delimited by the dotted rectangle in (J1–J4) is shown in (K1–K4) at higher magnification; note that some Y172-positive profiles were in close association with patches expressing VAChT (red, arrow). The data in (A,F) are shown as the mean ± SEM of 3–6 western blots from three independent cultures. Scale bars: E4,J4 = 20 μm (valid for C1-E3,H1–J3 , respectively); K4 = 4 μm (valid for K1–K3 ).

Article Snippet: The primary antibodies used were rabbit monoclonal anti-phospho-c-Jun (serine [Ser]63) clone Y172 (diluted 1:300, hereafter referred to as the Y172 antibody; Abcam, Cambridge, UK; cat. ab32385 or Millipore, Burlington, MA, USA; cat.# 04-212); rabbit polyclonal anti-phospho-c-Jun (Ser63; 1:100; Cell Signaling, Danvers, MA, USA; cat.# 9261); rabbit polyclonal anti-phospho-c-Jun (Ser73; 1:100; Cell Signaling; cat.# 9164); guinea pig polyclonal anti-synaptophysin 1 (1:500; Synaptic Systems, Goettingen, Germany; cat.# 101004); guinea pig polyclonal anti-vesicular acetylcholine transporter (VAChT; 1:500; Synaptic Systems, Goettingen, Germany; cat.# 139105); guinea pig polyclonal anti-vesicular glutamate transporter 1 (VGluT1, 1:500; Synaptic Systems, Goettingen, Germany; cat.# 135304); guinea pig polyclonal anti-vesicular GABA transporter (VGAT, 1:200; Synaptic Systems, Goettingen, Germany; cat.# 131004); mouse monoclonal anti-synaptic vesicle glycoprotein 2A (SV2, 1:1,000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# AB_2315386); mouse monoclonal anti-sigma-1 receptor (S1R, 1:50; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-137075); mouse monoclonal anti-Kv2.1 voltage-gated potassium channel (Kv2.1, 1:100; NeuroMab, Davis, CA, USA; cat.# 73-014); sheep polyclonal anti-choline acetyltransferase (ChAT, 1:1,000; Abcam cat.# Ab18736); rabbit polyclonal anti-ChAT (1:200; Millipore, Burlington, MA, USA; cat.# AB143); rabbit polyclonal anti-neuregulin-1 (NRG1) type III (extracellular, 1:250; Alomone labs, Jerusalem, Israel, cat.# ANR 113); mouse monoclonal anti-NRG-CRD, type III, clone N126B/31 (1:250; Millipore; cat.# MABN534); rabbit polyclonal anti-NRG1 1 α/β 1/2 (1:300; Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-348); mouse monoclonal anti-Golgi matrix protein of 130 kDa (GM130, 1:200; BD Biosciences, San Jose, CA, USA; cat.# 610822); mouse monoclonal anti-lysosomal membrane glycoprotein (LAMP-1), clone ID4B (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; cat.# ID4B); mouse monoclonal anti-KDEL (Lys-Asp-Glu-Leu motif) receptor (KDELR), clone KR-10 (1:50; Stressgen Biotechnologies, San Diego, CA, USA; cat.# VAA-PT048); mouse monoclonal anti-protein disulfide-isomerase (PDI), clone 1D3 (1:200; Enzo Life Sciences, Farmingdale, NY, USA; cat.# ADI-SPA-891); and mouse monoclonal anti-calcitonin gene-related peptide (CGRP; 1:100; Abcam, Cambridge, UK; cat.# ab81887).

Techniques: Western Blot, Cell Culture, Expressing, Staining, Immunostaining, Immunolabeling

Journal: Aging (Albany NY)

Article Title: Beneficial effects of dietary supplementation with green tea catechins and cocoa flavanols on aging-related regressive changes in the mouse neuromuscular system

doi: 10.18632/aging.203336

Figure Lengend Snippet: Effects of GTE- and cocoa-supplemented diets on excitatory cholinergic (VAChT-positive) and glutamatergic (VGluT1 and VGluT2) synaptic inputs to aged spinal MNs. ( A – F ) Graphs show the average density (number of puncta per 100 μm of MN soma perimeter, A , C , E ) and size (in μm 2 , B , D , F ) of the different types of afferent synapses examined; the red dashed line in each graph indicates the mean value of the corresponding afferent synapse density or size found in adult mice . ( G1 – O2 ) Representative confocal micrographs of VAChT, VGluT1 and VGluT2 nerve terminals contacting MN cell bodies of animals from control, GTE and cocoa groups, as indicated. Spinal cord sections were immunolabeled for either VAChT, VGluT1 or VGluT2 (green), and counterstained with fluorescent Nissl staining (blue) to visualize MN cell bodies, as indicated in panels. Data in the graphs are expressed as the mean ± SEM, ** p < 0.01 and *** p < 0.001 vs. control (one-way ANOVA, Bonferroni’s post hoc test); 50-60 MNs were analyzed per animal (number of animals per group: control =3, GTE = 4; and cocoa = 5). Scale bar in O2 = 20 μm (valid for G1 – O1 ).

Article Snippet: Vesicular acetylcholine transporter , VAChT , guinea pig polyclonal , Synaptic Systems (Gottingen, Germany / #139105 , 1:500.

Techniques: Control, Immunolabeling, Staining

Journal: Aging (Albany NY)

Article Title: Beneficial effects of dietary supplementation with green tea catechins and cocoa flavanols on aging-related regressive changes in the mouse neuromuscular system

doi: 10.18632/aging.203336

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry.

Article Snippet: Vesicular acetylcholine transporter , VAChT , guinea pig polyclonal , Synaptic Systems (Gottingen, Germany / #139105 , 1:500.

Techniques: Immunohistochemistry

Journal: Cell Death and Differentiation

Article Title: Kidins220/ARMS controls astrocyte calcium signaling and neuron–astrocyte communication

doi: 10.1038/s41418-019-0431-5

Figure Lengend Snippet: Kidins220 expression in astrocytes is required for proper neuronal development. a Wild-type neurons were plated on confluent wild-type (+/+) or Kidins220−/− astrocyte cultures, fixed and stained with anti-β tubulin III antibodies after 3 DIV to visualize, and quantify network development. Left: representative images of neuron–astrocyte cocultures. Scale bar, 25 µm. Middle: Sholl analysis of wild-type neurons grown on either wild-type or Kidins220−/− astrocytes for 3 DIV. Genotype effect: p = 0.03; **p < 0.01, repeated measures ANOVA followed by the Bonferroni’s multiple comparison test (n = 5 for both wild-type and Kidins220−/− cultures). Right: total dendritic length of wild-type neurons grown on either wild-type or Kidins220−/− astrocytes for 3 DIV. **p < 0.01, unpaired Student’s t test (n = 22 wild-type and Kidins220−/− cells from five independent cultures). Upper panels: representative images of wild-type neurons plated on confluent wild-type or Kidins220−/− astrocyte cultures stained with anti-VGLUT1 (b) or anti-VGAT (c) antibodies at 5, 7, and 10 DIV. Scale bars, 5 µm. Lower panels: quantification of the density of VGLUT1 and VGAT-positive boutons under the various experimental conditions. *p < 0.05, unpaired Student’s t test (n = 20 wild-type and Kidins220−/− cells from five independent cultures). Values are expressed as means ± S.E.M. in all panels

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Kidins220 (GSC16, #AB34790, Abcam, Cambridge, UK), rabbit monoclonal anti-GAPDH (14C10, #2118, Cell signaling, Leiden, The Netherlands), rabbit polyclonal anti-active caspase 3 (#AF835, R&D Systems, Minneapolis, MN, USA), rabbit anti-β tubulin III (#T2200, Sigma-Aldrich, Milan, Italy), guinea pig polyclonal anti-vesicular glutamate transporter-1 (VGLUT1, #AB5905, Merck-Millipore, Darmstadt, Germany), rabbit polyclonal anti-vesicular GABA transporter (VGAT, #131003, Synaptic System, Goettingen, Germany), mouse monoclonal anti-glial fibrillary acidic protein (GFAP, #G3893, Sigma-Aldrich), rabbit polyclonal anti-TRPV4 (#ab39260, Abcam), mouse anti-neuronal nuclei (NeuN, #MAB377, Merck-Millipore), chicken anti-NeuN (#266006, Synaptic Systems), guinea pig anti-Iba1 (#234004, Synaptic Systems), and rabbit anti-Olig2 (#AB9610, Merck-Millipore).

Techniques: Expressing, Staining