Multimodal Microscopy Imaging Protocol

Confocal laser scanning microscopy (CLSM) observations were conducted with an upright FV1000-D confocal laser scanning microscope (Olympus). For fluorescein diacetate (FDA) and propidium iodide (PI) staining, samples were soaked in dye solution containing 5 μg/ml FDA, 10 μg/ml PI, and 100 mM sorbitol. After 1 min, the samples were observed via CLSM. For calcofluor white staining, samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 60 min under a vacuum at room temperature. The fixed tissues were washed twice for 1 min in PBS and cleared with ClearSee (Kurihara et al., 2015 (link)). The cleared samples were stained with 0.1% calcofluor white in ClearSee solution for 60 min and then washed with ClearSee solution for 30 min. The stained samples were observed via CLSM. UPLSAPO10X or UPLSAPO60XW (Olympus) was used as the objective lens. The excitation wavelength and transmission range for emission were 473 nm and 485 to 560 nm for FDA and green fluorescent protein (GFP), 559 nm and 617 to 717 nm for PI, and 405 nm and 425 to 475 nm for calcofluor white.