Journal: eLife

Article Title: The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact

doi: 10.7554/eLife.91931

Figure Lengend Snippet: ( A, B ) Expanded synaptic contact areas labeled with anti-Cx35.5 (green) and anti-GluR2 (magenta) ( A : projection of 18 sections at 0.55 µm z-step size; B : projection of 15 sections at 0.50 µm z-step size). ( C ) ‘En face’ view of an expanded synaptic contact area showing that GluR2 labeling is restricted to the periphery of the contact, whereas Cx35.5 labeling is distributed throughout the whole contact area (projection of 46 sections at 0.65 µm z-step size). ( D ) Graph shows the lack of colocalization (see ‘Materials and methods’) between Cx35.5 and GluR2 fluorescence at individual club ending (CE) contacts, determined by the Manders’ colocalization coefficient: GluR2/Cx35.5 0.10 ± 0.020 (x-axis); Cx35.5/GluR2 0.08 ± 0.015 (y-axis), n = 13 CEs from five fish. Cross mark indicates the average value. ( E ) Quantification of fluorescence over area for Cx35.5 and GluR2 at the ‘Center” (central ¾) and ‘Periphery’ (remaining ¼) of the CE contact area. Values of fluorescence/area are represented as normalized to those of Cx35.5 in the center (higher value): GluR2 center: 0.25 ± 0.069; Cx35.5 periphery: 0.70 ± 0.126; GluR2 periphery: 0.96 ± 0.039 (n = 5 CEs from five fish). While fluorescence for Cx35.5 and GluR2 is not significantly different in the periphery (n.s.), Cx35.5 distinctly predominates over GluR2 at the center (Student’s t -test, p<0.0001). ( F ) Graphical description of the center vs. periphery distribution of Cx35.5 and GluR2 for the data described in ( E ). The scale bars represent actual dimensions; expanded images were not adjusted for expansion factor.

Article Snippet: The antibody mix, with block solution, included combinations of the following: rabbit anti-Cx35.5 ( , clone 12H5, 1:200), mouse IgG1 anti-Cx35/36, which labels both Cx35.5 and Cx35.1 (Millipore, Cat# MAB3045, 1:250), mouse IgG2A anti-Cx34.1 ( , clone 5C10A, 1:200), mouse IgG1 anti-ZO1 (Invitrogen, Cat# 33-9100, 1:200), mouse IgG1 anti-N-cadherin (BD Transduction Laboratories, Cat# 610920, 1:50), mouse IgG1 anti-beta-catenin (Sigma, Cat# C7207, 1:100), rabbit IgG anti-GluR2/3 (Millipore, Cat# 07-598, 1:200), and chicken IgY anti-GFP (Abcam, Cat# ab13970, 1:200).

Techniques: Labeling, Fluorescence

Journal: eLife

Article Title: The components of an electrical synapse as revealed by expansion microscopy of a single synaptic contact

doi: 10.7554/eLife.91931

Figure Lengend Snippet: ( A ) Double labeling with anti-Cx35.5 and anti-ZO1 shows a similar proportion of fluorescence at CEs (ZO1 45.24% ± 2.709; Cx35.5 41.53% ± 4.058; unlabeled 13.23%; n = 8 CEs from six fish). The region of interest (ROI) for analysis of fluorescence here and ( B ) was defined by the outline of Cx35.5 labeling of CEs. ( B ) Double labeling for N-cadherin and Cx35.5 (left), and for β-catenin and Cx35 (right) also shows similar proportionality (N-Cad 43.20% ± 3.334; Cx35.5 40.48% ± 3.041; unlabeled 16.32%; n = 13 from five fish; β-catenin 46.21% ± 2.728; Cx35.5 44.18% ± 1.671; unlabeled 9.61%; n = 13 CEs from seven fish). ( C ) Double labeling for Cx35.5 and GluR2 shows lack of proportionality, with Cx35.5 fluorescence occupying the majority of the CE contact area (GluR2 18.99% ± 1.601; Cx35.5 35.92% ± 2.087; unlabeled 45.10%; n = 10 CEs from five fish). ROI was defined in this case by the outline of GluR2 labeling. ( D ) Tree plot illustrating the area occupancy (fluorescence/contact area) for AJ (N-Cad = 38.1%), GJ (Cx35 = 34.8%), and glutamatergic (GluR2=18.9%) labeling at individual CEs (values normalized to ROI outlined by GluR2 labeling). The unlabeled area represents 8.1% of the contact’s surface. ( E ) The cartoon summarizes the synaptic components identified at a single CE contact. While chemical synapses are restricted to a small and peripheral area of the contact (presynaptic vesicles and release sites are represented in gray, postsynaptic receptor areas in magenta), most of its contact surface is occupied by multiple GJs (green) of variable size, which are interleaved and closely associated to AJs (red).

Article Snippet: The antibody mix, with block solution, included combinations of the following: rabbit anti-Cx35.5 ( , clone 12H5, 1:200), mouse IgG1 anti-Cx35/36, which labels both Cx35.5 and Cx35.1 (Millipore, Cat# MAB3045, 1:250), mouse IgG2A anti-Cx34.1 ( , clone 5C10A, 1:200), mouse IgG1 anti-ZO1 (Invitrogen, Cat# 33-9100, 1:200), mouse IgG1 anti-N-cadherin (BD Transduction Laboratories, Cat# 610920, 1:50), mouse IgG1 anti-beta-catenin (Sigma, Cat# C7207, 1:100), rabbit IgG anti-GluR2/3 (Millipore, Cat# 07-598, 1:200), and chicken IgY anti-GFP (Abcam, Cat# ab13970, 1:200).

Techniques: Labeling, Fluorescence

Journal: Biomolecules

Article Title: Functional, Morphological and Molecular Changes Reveal the Mechanisms Associated with Age-Related Vestibular Loss

doi: 10.3390/biom13091429

Figure Lengend Snippet: Age-related changes in the numbers of pre- and postsynaptic synaptic elements in the vestibular sensory epithelia between young (1.5-month-old) and old (24-month-old) mice. ( A ). Age-related changes in the numbers of pre-synaptic ribbons and postsynaptic glutamate receptor patches were investigated using immunofluorescence in the striolar region of the utricular macula. Sensory epithelia were immunostained with antibodies against CTBP2 (green) to identify presynaptic ribbons and GluR2/3 (red) to identify postsynaptic glutamate receptor patches (red). ( B ). There was no significant difference in the density of presynaptic ribbons (CTBP2). ( C ). There was no significant difference in the density of postsynaptic glutamate receptor patches (GluR2/3). Non-significant differences are indicated by n.s.

Article Snippet: The first combination included mouse monoclonal (IgG1) anti-CTBP2 (BD Biosciences 612044; RRID: AB_399431, San Jose, CA, USA) and rabbit polyclonal anti-GluR2/3 (Millipore AB1506; RRID: AB_90710, Burlington, MA, USA).

Techniques: Immunofluorescence