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)).
Three-colour STED: Due to incompatible species specificities of primary antibodies available (for RINT1, TANGO1 and ERGIC-53), we were forced to use sub-optimal secondary antibodies. We used Alexa 488, Alexa 594 and Alexa 647. This required that we set the depletion laser (775 nm) at only 3–8% intensity for the Alexa 647 channel to prevent rapid bleaching.
Three-colour STED: Due to incompatible species specificities of primary antibodies available (for RINT1, TANGO1 and ERGIC-53), we were forced to use sub-optimal secondary antibodies. We used Alexa 488, Alexa 594 and Alexa 647. This required that we set the depletion laser (775 nm) at only 3–8% intensity for the Alexa 647 channel to prevent rapid bleaching.
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