Confocal imaging was performed on a LSM780NLO confocal system fitted on an Observer Z1 inverted microscope (Zeiss) equipped with a Chameleon Vision II 690-1064 nm multiphoton laser (Coherent Europe). Fluorochromes were separated by linear unmixing using ZEN 2012 software (Zeiss). IUE were imaged using a Plan Apochromat 20x/0,8 dry objective, in vivo cilia imaging was done using LD C Apochromat 40x/1.1 water immersion objective and in vitro cilia imaging was done using LD C Apochromat 63x/1.4 oil immersion objective (Zeiss). Wholemount cortices were flattened into fluorodish (Ibidi) and covered with glass coverslip before image acquisition using the 63x objective on 4-5 Z-stacks spaced at 0.3 μm to cover the whole tight-junction region.Images were then processed using the Fiji/ImageJ software (Schindelin et al., 2012 (link)).
Sted images were acquired using a DMi8 inverted microscope (Leica) equipped with STED with 592nm (CW), 660nm (CW) and 775nm (pulsed) depletion lasers. HC PL APO 100x/1.4 oil objective was used. Acquisition was done with LAS X and deconvolution using Huygens Professional Version 17.1. For STED imaging, ABBERIOR secondary antibodies were used.
Sted images were acquired using a DMi8 inverted microscope (Leica) equipped with STED with 592nm (CW), 660nm (CW) and 775nm (pulsed) depletion lasers. HC PL APO 100x/1.4 oil objective was used. Acquisition was done with LAS X and deconvolution using Huygens Professional Version 17.1. For STED imaging, ABBERIOR secondary antibodies were used.