Fig. 1: Overview of enGLOW processing and specialized equipment.
From: enGLOW 3D microscopy of the enteric nervous system in cleared human and mouse gut
a Diagram describing the steps to process mouse and human tissues. Prior to enGLOW processing, antibody validation is required on cryosections (steps A–J). Sample preparation is relevant for mouse GI tissues as they are obtained fresh while human samples are received pre-fixed (steps 1–8). Both human and mouse samples then start pre-treatment which involves delipidation and background fluorescence bleaching (steps 9 to 13). Immunolabeling enables the tagging of proteins of interest with fluorescent reporters that will be imaged in lightsheet fluorescence microscopy (steps 14–19). Cleared samples (steps 20–23) are then mounted for imaging (step 24). Processing mouse samples takes 2 weeks from start to finish, while human samples take 3 weeks due to prolonged immunolabeling incubations. Actual bench time is approximately 3 h for both sample types. b Diagram with dimensions of PTFE box used to protect human tissues during enGLOW processing. c Diagram with dimensions of custom holder used to image cylindrical mouse samples. Abbreviations: RT room temperature, PBS phosphate buffered saline, PFA paraformaldehyde, DCM dichloromethane, DBE dibenzyl ether. Created in BioRender. Planchette, A. (2025) https://BioRender.com/165ygdm.
