guinea pig polyclonal anti-synaptophysin  (Synaptic Systems)

Journal: bioRxiv

Article Title: Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model

doi: 10.1101/2024.09.05.611287

Figure Lengend Snippet: A . Widefield fluorescence image of cultured neurons on top of the nlgn micropattern (gray) fixed at 15 div, labeled for adducin (purple), bassoon (cyan) and map2 (orange). B . Zooms corresponding to the areas highlighted in A. Top row: natural synapses (NS) at axo-dendritic contacts; middle row: induced presynapses at isolated axon-micropattern dots (S+); bottom row: isolated axon with no induced presynapse (S-). C . Widefield fluorescence image of cultured neurons on top of the nlgn micropattern (gray) fixed at 15 div, labeled for β2-spectrin (purple), synaptophysin (cyan), and map2 (orange). D . Zooms corresponding to the NS, S+ and S-highlighted in C. E . Widefield fluorescence image of cultured neurons on top of the nlgn micropattern (gray) fixed at div 15, labeled for actin (purple), feeding with anti-synaptotagmin antibody (syt) during constitutive vesicular cycling (cyan) and map2 (orange). F . Zooms corresponding to the NS, S+, and S-highlighted in E. Scale bars for A, C, E: 20 µm. Scale bars for B, D, F: 10 µm. G . Proportion of the length of isolated axons showing presynaptic specializations following the nlgn micropattern (patterned axon) over the total length of isolated axons selected within the experimental conditions (bassoon, synaptophysin, synaptotagmin) compared to controls (pooled labels, see ). H . Mean intensity for bassoon (dark cyan, N=2; n>100), synaptophysin (light green, N=4; n>200), synaptotagmin (pink; N=5, n>200) and actin (blue) at natural synapses (NS), induced presynapses (S+), and isolated axon devoid of presynapse (S-), normalized to natural synapses.

Article Snippet: Primary antibodies used are chicken anti-map2 (1:1,000, ab5392, RRID:AB_2138153; Abcam), mouse anti-ß2-spectrin (1:100, clone 42/aa 2101-2189, #612563, RRID:AB_399854; BD Bioscience), guinea pig anti-synaptophysin (1:400, 101 004, RRID:AB_1210382; Synaptic Systems), mouse anti-synaptotag-min1 (1:600, clone 604.2/aa 1-12, 105 311, RRID:AB_993036; Synaptic Systems), rabbit anti-adducin alpha (1:100, ab40760, RRID:AB_722627; Abcam), mouse anti-bassoon (1:200, clone SAP7F407, ab82958, RRID:AB_1860018; Abcam), rabbit anti-GFP (1:750, ab290, RRID:AB_303395; Abcam), mouse anti-GFP (1:750, clone 9F9.F9, ab1218, RRID:AB_298911; Abcam), rabbit anti-munc13-1 (1:500, 126 103, RRID:AB_887733; Synaptic Systems).

Techniques: Fluorescence, Cell Culture, Labeling, Isolation

Journal: bioRxiv

Article Title: Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model

doi: 10.1101/2024.09.05.611287

Figure Lengend Snippet: A . Widefield fluorescence image of cultured neurons on top of the Fc-BSA micropattern (top row, gray) or outside of the pattern (bottom row) fixed at div 15 labeled for adducin (purple), bassoon (cyan), map2 (orange). Scale bar, 10 µm. B . Proportion of the length of isolated axons showing presynaptic bassoon specializations following the nlgn micropattern (patterned axon) over the total length of isolated axons selected within the experimental conditions (nlgn dots, N=5) compared to controls (Fc-BSA, N=3; outside of the nlgn dots pattern, N=4). C . Widefield fluorescence image of cultured neurons on top of the Fc-BSA micropattern (top row, gray) or outside of the pattern (bottom row) fixed at div 15 labeled for ß2-spectrin (purple), synaptophysin (cyan), map2 (orange). Scale bar, 10 µm. D . Proportion of the length of isolated axons showing presynaptic synaptophysin specializations following the nlgn micropattern (patterned axon) over the total length of isolated axons selected within the experimental conditions (nlgn dots, N=4) compared to controls (Fc-BSA dots, N=3; outside of the nlgn dots pattern, N=4).

Article Snippet: Primary antibodies used are chicken anti-map2 (1:1,000, ab5392, RRID:AB_2138153; Abcam), mouse anti-ß2-spectrin (1:100, clone 42/aa 2101-2189, #612563, RRID:AB_399854; BD Bioscience), guinea pig anti-synaptophysin (1:400, 101 004, RRID:AB_1210382; Synaptic Systems), mouse anti-synaptotag-min1 (1:600, clone 604.2/aa 1-12, 105 311, RRID:AB_993036; Synaptic Systems), rabbit anti-adducin alpha (1:100, ab40760, RRID:AB_722627; Abcam), mouse anti-bassoon (1:200, clone SAP7F407, ab82958, RRID:AB_1860018; Abcam), rabbit anti-GFP (1:750, ab290, RRID:AB_303395; Abcam), mouse anti-GFP (1:750, clone 9F9.F9, ab1218, RRID:AB_298911; Abcam), rabbit anti-munc13-1 (1:500, 126 103, RRID:AB_887733; Synaptic Systems).

Techniques: Fluorescence, Cell Culture, Labeling, Isolation

Journal: bioRxiv

Article Title: Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model

doi: 10.1101/2024.09.05.611287

Figure Lengend Snippet: A . First to third images, widefield fluorescence image of cultured neurons on top of the nlgn micropattern (green) fixed at 15 div, labeled for map2 (purple), synaptophysin (orange) and actin (gray). Scale bar, 5 µm (first image), 1 µm (second and third images, zooms of the area highlighted on the first image). Fourth image shows the nlgn pattern (green) superimposed with the PAINT image of the bassoon staining (orange) and the STORM image of the actin staining (gray). Scale bar, 1 µm. Fifth image is a zoom of the area highlighted on the fourth image showing the micropattern (green shape), bassoon clusters from the PAINT image (orange contours), and colorized presynaptic actin nano-structures from the STORM image (gray, corral in blue, mesh in red, rails in green). Scale bar, 500 nm. B . Zooms of the presynapse shown in A showing the STORM image of actin (gray) and PAINT image of bassoon (orange) in XY (left column) and transverse (XZ with a vertical Z axis, right column) views. The transverse view is taken along the bracket shown on the XY view. Scale bars, 500 nm (XY views), 200 nm (transverse XZ views). C . ChimeraX renderings corresponding to the views shown in B with XY (left column) and transverse (right column) views of actin nanostructures (gray) and bassoon nanoclusters (orange). The transverse XZ views show how actin is present above and in-between bassoon nanoclusters.

Article Snippet: Primary antibodies used are chicken anti-map2 (1:1,000, ab5392, RRID:AB_2138153; Abcam), mouse anti-ß2-spectrin (1:100, clone 42/aa 2101-2189, #612563, RRID:AB_399854; BD Bioscience), guinea pig anti-synaptophysin (1:400, 101 004, RRID:AB_1210382; Synaptic Systems), mouse anti-synaptotag-min1 (1:600, clone 604.2/aa 1-12, 105 311, RRID:AB_993036; Synaptic Systems), rabbit anti-adducin alpha (1:100, ab40760, RRID:AB_722627; Abcam), mouse anti-bassoon (1:200, clone SAP7F407, ab82958, RRID:AB_1860018; Abcam), rabbit anti-GFP (1:750, ab290, RRID:AB_303395; Abcam), mouse anti-GFP (1:750, clone 9F9.F9, ab1218, RRID:AB_298911; Abcam), rabbit anti-munc13-1 (1:500, 126 103, RRID:AB_887733; Synaptic Systems).

Techniques: Fluorescence, Cell Culture, Labeling, Staining