Echo revolution microscope

H₂O₂ was detected in roots using the 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) staining method. The roots from 7-day-old seedlings were incubated in H₂O containing 50 µM H₂DCFDA for 30 min under dark conditions. Then, the roots were rinsed with sterilized water three times. The fluorescent signals were observed with an Echo Revolution microscope (Echo). The excitation and emission wavelengths used for detection of the signals were 488 nm and 520 nm, respectively.
To detect superoxide, seedlings were stained for 2 min in a solution of 2.5 mM Nitroblue tetrazolium (NBT) in 50 mM phosphate buffer (pH 6.1) in the dark and then rinsed three times with distilled water^{49 (link)}. Images for NBT staining were obtained using a 1× objective with a Nikon SMZ1270 Stereo microscope.

Tissue samples were fixed in 10% formalin while organoids had their media removed and 500 µL of Formalin-acetic acid-alcohol (FAA, composition in ref. ¹⁸ ) was added to wells containing Matrigel. Both tissues and organoids were changed to 70% ethanol 24 h later prior to paraffin-embedding. Tissue and organoids were stained with hematoxylin and eosin (H&E), Alcian Blue, and Periodic acid–Schiff (PAS) at the Iowa State University Histopathology Department. After staining, slides were scanned on Leica Aperio GT 450 Scanner and analyzed with ImageScope (v12.4.3.5008) to characterize the morphology and cell type composition of tissues and organoids. For immunohistochemistry, slides were stained for proliferating cell nuclear antigen (PCNA) (DAKO; 0879) at a 1:400 dilution, and images were taken on an ECHO Revolution microscope (ECHO).