Fixing the Disks and Confocal Microscopy imaging
The Disks containing cells were initially cut to half or small pieces and transferred onto a 6-well plate and then fixed using a 4% paraformaldehyde solution for 24 hours. Following the fixation, the paraformaldehyde solution was removed, and the disks were washed three times with PBS. The Disks were subsequently imaged and processed using a CellVoyager CV8000 Yokogawa confocal scanner microscope. For imaging, a z-depth range of -150μm to 550μm was selected, with a scanner layer thickness of 6.5μm. The lasers used were 640nm for PCF labeled with Texas Red and 488nm for EEC labeled with GFP. The objective lens chosen for observing the Purkinjean was the 4x lens. The acquired images were then merged and analyzed using CellPathfinder (a high-content analysis software developed by Yokogawa) to generate 2D or 3D aligned images.