Nunc lab tek 2 chambered coverglasses

Fixed and stained organoids were imaged in Nunc™ Lab-Tek™ II Chambered Coverglasses (Thermoscientific^TM # 155360) using Nikon A1 HD25/A1R HD25 confocal microscope equipped with NIS-Elements controller. 20X and 40X water immersion objectives were used to take z-stack images. To quantify organoid size and number, merged maximum intensity projections (DAPI and Phalloidin) of large images were processed in Fiji software as follows: image > type > 16-bit > adjust threshold > analyze particles. Fiji presents then the count and the size of all areas in the image. Each area represents an organoid.

Exosomes were purified from the conditioned media using Invitrogen Total Exosome Isolation Reagent (from cell culture media; Cat#: 4478359, ThermoFisher) according to the manufacturer’s instructions. The explants or SGNs were cultured in 400 μl SGN basic medium in Nunc Lab-Tek II Chambered Coverglasses (Cat#: 155409, ThermoFisher) with exosomes (5 μg/ml), with or without SU5408 (100 nM for SGN explant and 50 nM for SGNs) for five continuous days. Media was changed every 2 d.

Organoids were embedded in Matrigel and grown on Nunc™ Lab-Tek™ II Chambered Coverglasses (Thermoscientific^TM # 155360) for the indicated days. After cold PBS wash on ice (10 min), organoids were fixed with cold 4% paraformaldehyde (at 4°c for 20–25 min). Cold PFA causes Matrigel meltdown allowing organoids to be fixed to the bottom of Chambered Coverglasses. After 1h permeabilization with 0.5% cold triton and 2-3h blocking with 1.5% BSA solution containing 0.1% triton, organoids were incubated with the primary antibodies in the blocking solution overnight at 4°C. The day after, organoids were washed three times with PBS and incubated with secondary antibodies coupled with Alexa Fluor-488 at 0.5 μg/ml concentration for 2-3h. DNA was stained with DAPI and F-actin with Phalloidin (1/500). Organoids were PBS washed, then imaged in the imaging solution (0.7 mM N-acetyl cysteine, PH~7.4).