Hxp 120v fluorescent lamp

The images of organotypic explant cultures were captured using a Zeiss Imager Z.2 fluorescence microscope, equipped with ApoTome 2, an Axiocam 506 mono camera, and HXP-120V fluorescent lamp (Carl Zeiss Microscopy, Oberkochen, Germany). The excitation (λExc.)/emission (λEm.) characteristics of the filter sets used for the different fluorophores were as follows (in nm): DAPI (λExc. = 369 nm, λEm = 465 nm), AF488 (λExc. = 490 nm, λEm = 525 nm), AF568 (λExc. = 578 nm, λEm = 602 nm), and ToPro (λExc. = 642 nm, λEm = 661 nm). The Zen 2.3 blue edition software (Zeiss) captured images (tiled and z-stack, 20× magnification). Sections of 12-µm thickness were analyzed using 12–15 Apotome Z-planes. For the quantification of positive cells in the retinal ONL, we proceeded as follows: The number of cells in six different rectangular ONL areas was counted manually based on the number of DAPI-stained nuclei and used to calculate an average ONL cell size. This average ONL cell size was then used to rapidly calculate the total number of cells in a given ONL area. The percentage of positive cells was calculated by dividing the absolute number of positive cells by the total number of ONL cells.

Immunohistochemical sections were imaged on a Zeiss Imager Z.2 fluorescence microscope, equipped with ApoTome 2, an Axiocam 506 mono camera and an HXP-120V fluorescent lamp (Carl Zeiss Microscopy, Oberkochen, Germany). The ZEN 3.3 (blue edition) software (Carl Zeiss Microscopy) captured z-stack images using 20x and 40x magnifications. The quantification of the rod/cone OS length, cone density and the number of outer nuclear layers (ONLs) was done by averaging measurements from at least four sections per animal. Per section, three distinct measurements were taken and averaged (Roche et al., 2016 (link)). Dorsal and ventral sections taken approx. 300 μm from the center of the visual streak were considered as peripheral retina. Figures were prepared using Photoshop CS5 (Adobe, San Jose, CA, USA). OCT reflectivity profiles between the upper ganglion cell layer and the bottom retinal pigment epithelium (RPE) layer were generated and analyzed using the ImageJ software package (NIH, Bethesda, MD, USA) as described previously (Garcia Garrido et al., 2014 (link)).