Cyanine3 (cy3)

Periodic acid–Schiff diastase staining was graded qualitatively, based on the morphological classification system described by von Herbay (16 (link)) and colleagues and semi quantitatively as described by Baisden et al. (16 (link), 29 (link)).
The fluorescence slides were analyzed using an Olympus BX 51 epifluorescence microscope equipped with filters sensitive for DAPI (Excitation peak: 358 nm, DNA-emission peak: 461 nm and RNA-emission peak: 500 nm), Cy3 (Excitation peak: 550 nm, Emission peak: 570 nm), and Cy2 (Excitation peak: 492 nm, Emission peak: 510 nm) (Olympus corporation, Japan).
Photos were taken with a digital microscope (Keyence BZ9000 E, Osaka, Japan). Representative microphotographs were taken with filters for the detection of Cy3 (OP-66838 BZ TRITC Excitation wavelength: 540/25 Dichroic mirror wavelength: 565 Absorption wavelength: 605/55), Cy2 (OP-66836 BZ filter GFP-BP Excitation wavelength 470/40 Dichroic mirror wavelength: 495 Absorption wavelength: 535/50), and DAPI (OP-66834 BZ filter DAPI-BP Excitation wavelength 360/40 Dichroic mirror wavelength: 400 Absorption wavelength 460/50). Microphotographs were processed using the GNU Image Manipulation Program (GIMP, Version 2.8) on an IBM compatible PC running Windows 10.

Images were acquired using an inverted Olympus IX81 microscope equipped with an Olympus FV1000 confocal laser scanning system, a FVD10 SPD spectral detector and diode lasers of 495 nm (Alexa488) and 550 nm (Cy3) (Olympus, Tokyo, Japan). All images shown were acquired with an Olympus UPLSAPO 60x (oil, numerical aperture: 1.35) objective. The images were further developed and organized by Adobe Photoshop (Adobe, San Jose, CA, United States) or ImageJ (1.51)/Fiji¹.