Confocal microscopy

Fluorescent confocal images of PNEC/NEB cells immunostained for SYP, Mash1, Prox1, Ki67, and HIF-1alpha in double/triple-immunostained sections were obtained using a Leica confocal laser scanning microscope (model TCS-SPE) and LAS-AF software (Leica Microsystems, Wetzlar, Germany), as previously reported.²² The variable excitation wavelengths of the krypton/argon laser were 488 nm for fluorescein isothiocyanate conjugate, 568 nm for Texas Red complex, and 695 nm for Alexa Fluor 680 conjugate/RedDot 2 (nuclear counterstaining). Image processing, including color resolution, color separation, and merging of fields, was carried out using Adobe PhotoShop CS2 software (Adobe Systems Incorporated, San Jose, CA, USA). To visualize these images in different color from before and after the stripping/re-probing protocol, pseudo-color was applied with the channel option of Adobe Photoshop CS3 (Adobe Systems Incorporated).