Imager m2 upright microscope

Cultured neurons and astrocytes were infected with small-scale AAVs at DIV14. After 3 days, these cells were treated with 500 μM biotin for 6h. Neurons and astrocytes were fixed at indicated time points in 4% PFA/ 4% sucrose for 20 min at room temperature. They were permeabilized with 0.1 % Triton-X 100 and 10% normal goat serum (NGS) for 30 min at room temperature. Samples were then incubated for overnight at 4 °C with primary antibodies followed by Alexa Fluor 488-, Alexa Fluor 555 or Alexa 647-conjugated secondary antibodies diluted in PBS containing 0.01% Triton X-100 and 10% NGS for 2 h at room temperature. The neuron and HEK293T cells mixed-culture assay was performed as previously described^{32 (link),33 (link)}. Briefly, HEK293T cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions. After 20 h, transfected HEK cells were seeded on cultured neuron at DIV14. Fluorescence images were acquired with Zeiss Imager M2 upright microscope equipped with an Apotome module, Zeiss 710, Zeiss 780, Zeiss 880 confocal microscopes using the Zen Software or a stimulated emission depletion (STED) super resolution microscope (TCS SP8 STED, Lecia Microsystems) using the Leica Application Suite (LAS) software. The individual acquiring the images was always blinded to the experiment. Images were quantified and post-processed using FIJI.