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. 2021 Mar 22;10:e64527. doi: 10.7554/eLife.64527

Figure 9. Syt-1 and Syt-7 displays limited colocalization.

(A) Single confocal slices of new-born mouse chromaffin cells stained against Syt-1 (α-Syt-1) and Syt-7 (α-Syt-7) in WT cells and in Syt-7 KO cells, and merged images. Scale bar: 5 µm. (B) Quantification of staining against Syt-1 and Syt-7 in WT and Syt-7 KO cells. For staining with other antibodies: see Figure 9—figure supplement 1D–F. (C) Manders’ coefficients M1 and M2 (mean ± SEM) for co-localization analysis of Syt-1 and Syt-7 in WT and Syt-7 KO cells. (D) Single optical slices of new-born WT mouse chromaffin cells stained against Syt-1 (α-Syt-1) and Syt-7 (α-Syt-7) acquired with 3D-structured illumination microscopy (3D-SIM). Scale bar: 2 µm. Bottom right: Magnified ROIs. ROI 1: A section of the cell from the merged-channel image showing that the majority of the spots are identified either as Syt-1 -or Syt-7-positive and few are positive for both isoforms. Scale bar: 1 µm. ROI 2: An example of a spot where Syt-1 and Syt-7 appear co-localized (top panel: α-Syt-1; middle panel: α-Syt-7; bottom panel: A merged image of α-Syt-1 and α-Syt-7 channels). Scale bar: 0.2 µm. (E) Quantification of the percentage of Syt-1 spots that were costained for Syt-7 and vice versa. (F) Number of Syt-1 and Syt-7 spots per cell, while analyzing every third optical slice (thickness of each 0.11 µm) from the top to the bottom of the cells using the ComDet plugin for ImageJ. (G) The total number of spots per cell. Bar colors indicate the proportions of Syt-1 (cyan), Syt-7 (red) and Syt-1/Syt-7 (white) spots. (H) Number of Syt-1/Syt-7 spots where the two isoforms are considered to be colocalized to the same vesicle as shown in ROI two example (D). Data information: Values are mean ± SEM. *: p<0.05; ****: p<0.0001, Student’s t-test. Number of cells in (B, C): Syt-7 WT: N = 22 cells; Syt-7 KO: N = 22 cells. Number of cells in (E–H): Syt-7 WT, N = 5 cells.

Figure 9.

Figure 9—figure supplement 1. Syt-1 and Syt-7 limited colocalization.

Figure 9—figure supplement 1.

(A–C) Immunostaining and quantification of Syt-1 and Syt-7 expression in the absence of glutaraldehyde during the fixation step. Antibodies are a α-Syt-7 rabbit polyclonal antibody and a α-Syt-1 mouse monoclonal antibody (105173 and 105011 from Synaptic Systems; see Materials and methods). (A) Single confocal slices of newborn mouse chromaffin cells stained against Syt-1 (α-Syt-1) and Syt-7 (α-Syt-7) in WT cells and in Syt-7 KO cells, and merged images. Scale bar: 5 μm. (B) Quantification of Syt-1 and Syt-7 staining in WT and Syt-7 KO cells. Some residual unspecific staining was detected in the Syt-7 KO cells. (C) Manders’ coefficients (mean ± SEM) for the colocalization analysis of Syt-1 fraction in Syt-7 and Syt-7 fraction in Syt-1 in WT and Syt-7 KO cells. (D–F) Immunostaining and quantification of Syt-1 and Syt-7 expression using a mouse monoclonal Syt-7 antibody (clone 275/14, MABN665, Sigma-Aldrich) and a rabbit polyclonal Syt-1 antibody (W855; T.C. Südhof). (D) Single confocal slices of newborn mouse chromaffin cells stained against Syt-1 (α-Syt-1) and Syt-7 (α-Syt-7) in WT cells and in Syt-7 KO cells, and merged images. Scale bar: 5 μm. (E) Quantification of the staining against Syt-1 and Syt-7 in WT and Syt-7 KO cells. (F) Manders’ coefficients (mean ± SEM) for the colocalization analysis of Syt-1 fraction in Syt-7 and Syt-7 fraction in Syt-1 in WT and Syt-7 KO cells. (G) Protein immunoblot probed with antibodies against Syt-7 (α-Syt-7) and VCP (α-VCP) as a loading control in Syt-7 KO or WT adrenal glands and brains obtained from three new-born mice (one lane per animal).