STED Imaging of TANGO1 Localization

STED images were taken on a TCS SP8 STED 3 × microscope (Leica Microsystems) on a DMI8 stand using a 100 × 1.4 NA oil HCS2 PL APO objective and a pulsed supercontinuum light source (white light laser). Images were acquired and deconvolved exactly as described before (Raote et al., 2017 (link); Raote et al., 2018 (link)). In all images acquired and presented here, a goat-anti mouse secondary antibody, coupled to Abberior 635 was used to visualise anti-HA antibody localisation, while secondary antibody coupled to Alexa 594 was used to visualise the antibody targeted against the lumenal epitope of TANGO1. Axial resolution were found to be ∼60 nm for the Abberior 635 channel and 50 nm for the Alexa Fluor 594 channel as described before (Raote et al., 2017 (link)).

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Raote I., Ernst A.M., Campelo F., Rothman J.E., Pincet F, & Malhotra V. (2020). TANGO1 membrane helices create a lipid diffusion barrier at curved membranes. eLife, 9, e57822.

Publication 2020

TCS SP8 STED 3 × microscope oil HCS2 PL APO objective

Alexa 594 Anti antibody Antibody Epitope Goat Light Microscope Mouse

Corresponding Organization :

Other organizations : Centre for Genomic Regulation, Yale University, Institute of Photonic Sciences, Université Paris Cité, Université Paris Sciences et Lettres, École Normale Supérieure - PSL, Sorbonne Université, Centre National de la Recherche Scientifique, Pompeu Fabra University, Institució Catalana de Recerca i Estudis Avançats

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Variable analysis

independent variables

Type of microscope used (TCS SP8 STED 3 × microscope (Leica Microsystems))
Objective lens used (100 × 1.4 NA oil HCS2 PL APO objective)
Light source used (pulsed supercontinuum light source (white light laser))

dependent variables

Localization of anti-HA antibody (visualized using goat-anti mouse secondary antibody coupled to Abberior 635)
Localization of antibody targeted against the lumenal epitope of TANGO1 (visualized using secondary antibody coupled to Alexa Fluor 594)

control variables

Deconvolution method (as described in Raote et al., 2017 and Raote et al., 2018)
Axial resolution of Abberior 635 channel (∼60 nm)
Axial resolution of Alexa Fluor 594 channel (∼50 nm)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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