Phenix high content screening system

Organoids were fixed in 4% paraformaldehyde (Alfa Aesar, no. J61899-AP), cryoprotected in 30% sucrose solution overnight, embedded in optimum cutting temperature (OCT) compound (VWR, no. 25608-930), flash frozen in dry ice with ethanol, and kept at −80^oC overnight. Organoids were cryosectioned (Leica CM1950 Clinical Cryostat) at 6 µm and mounted on Micro Slides, Superfrost^TM Plus (VWR, 48311-703). Slides were washed with PBS (one time, 5 min), blocked for 20 min (5% normal donkey serum, 1.5% Tween-20), and incubated overnight at 4 °C with primary antibody (in blocking buffer). Later the slides were washed with PBS (three times, 10 min each) and incubated with secondary antibody (PBS, 1.5% Tween-20) for 2 h at room temperature. The slides were then washed with PBS (one time, 10 min). For phalloidin staining, the slides were incubated with Phalloidin-647 for 20 min at room temperature and washed with PBS (one time, 10 min). The sections were stained with DAPI for 5 min, washed with PBS (three times, 10 min), and mounted using ProLong^TM Gold antifade reagent. Images were obtained by confocal microscopy (PerkinElmer Opera Phenix High Content Screening System).

Neurons grown on μ-Plate 96 well (Ibidi) were imaged for fluorescent pHrodo-labeled protein (excitation at 561 nm and emission at 570–630 nm) in an acidic environment using a 40× confocal Opera Phenix high-content screening system (PerkinElmer). Bright-field and fluorescence emission images were collected at 10-min intervals. Parameters for fluorescent objects were set (fluorescent intensity and contrast) and quantified using the Harmony software (PerkinElmer). Data were analyzed using Prism Software (GraphPad). Typically, conditions were repeated at least in triplicate and <4 (typically 8) fields were recorded per well. To track individual objects, images were collected at 1-s intervals. Images were analyzed in Harmony 4.5 (PerkinElmer). Densely clumped areas of wells were excluded, and a spot-finding algorithm was used to detect foci of pHrodo-labeled protein. Within the spots, fluorescent intensity of the Alexa-568 channel was quantified representing the amount of labeled protein taken up into the endosomes. A high-intensity threshold was used with the spot-finding algorithm to detect only the very bright spots within the wells; discrete spots were counted to determine the number of pHrodo-positive vesicles within the cells.