Open Access Highly Accessed Methodology article

Studying synaptic efficiency by post-hoc immunolabelling

Jorge Ramírez-Franco, Beatris Alonso, David Bartolomé-Martín, José Sánchez-Prieto* and Magdalena Torres*

Author Affiliations

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid 28040, Spain

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BMC Neuroscience 2013, 14:127  doi:10.1186/1471-2202-14-127

Published: 18 October 2013

Additional files

Additional file 1: Figure S1:

Serial reconstruction of the imaged field. A) Synaptic boutons loaded with FM1-43 dye before (to) and after (tend) stimulation with potassium chloride; contrast phase images of the same experiment field (white box) at different magnifications: 60× (B), 40 × (C) and 20× (D). E) Serial reconstruction of 20× cell phase images of the field monitorized during the experiment (white box) and surrounding areas. F) Contrast phase image al 20× magnification showing the field where the experiment was performed after fixing and labelling with the different antibody (matching field). Note the presence of distinctive hallmarks.

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Additional file 2: Figure S2:

Western blot of RIM1α and Munc13-1 confirmed specificity of both antibodies. A) Western Blot of Munc13-1 (top green), β-Tubulin (bottom, red) and synaptophysin (bottom, green), showing a single band for Munc13-1 with the expected molecular weight. B) Western blot of RIM1α (green, top) and β-Tubulin (green, bottom) showing that both antibodies recognize a single band with the expected molecular weight.

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Additional file 3:

Supplementary methods.

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Additional file 4: Figure S3:

FM1-43 experiment analysis routine using IgorPro software and ImageJ. A) Parameters used in IgorPro interface to automatically analyze the FM1-43 experiment. This software renders the mask and a set of background subtracted images. B) Import of the QualitySegment.ibw file in ImageJ software to draw a binary mask. The size of the image has to fit with the original images acquired during the experiment. C) Generation of the ROIset with ImageJ software by analyzing particles present in the mask. D) Import of the set of background-subtracted images for further analysis of the experiment with OriginPro.

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Additional file 5: Figure S4:

Alignment of FM1-43 with IR puncta after post-hoc ICC. A) Immunocytochemical images of FM1-43 in blue (pseudo colour), Alexa 488 (green) and Alexa 594 (red). Upper panels show ROIset superimposition and lower panels show line plot along a fiber. Note that the different ROIs are well fitted to the puncta in the different channels, this step is useful to visually check the alignment of the three channels. B) Arbitrary fluorescence units plot over the three channels of the ROIs indicated in A).

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