Zen blue edition software

Samples were cytospun on microscopic slides using a StatSpin Cytofuge 2 (1000 rpm, 2 min) and left to dry overnight. Slides were stained with May-Grünwald stain (50% working solution, 5 min) and, subsequently, Giemsa stain (10% working solution, 20 min). Morphology was examined using AxioVert 200 microscope and images were photographed using Axiocam MRc 5 camera and ZEN software, blue edition (Carl Zeiss AG, Oberkochen Germany).

Quantification of proliferating and differentiated cells was performed on 5 random fields of view (FOVs) on each of the two stained coverslips, making a total of 10 FOVs per condition. FOVs were determined in the DAPI channel. The images were acquired at ×200 magnification, capturing an average of 120 cells per FOV, using a Zeiss AxioImager Z1 microscope and Zeiss ZEN software (blue edition). BrdU⁺, β-III-tubulin⁺, GFAP⁺ and DAPI⁺ cells were counted using Adobe Photoshop®. The proportion of proliferating or differentiated cells per experiment was determined by quantifying the number of BrdU⁺ cells or β-III-tubulin⁺ and GFAP⁺ cells relative to the total number of cells (DAPI⁺ cells), with one data point referring to the average value of 10 FOVs. A total of ~1200 cells per condition were analysed.

One-μm-thick cross-sections of the optic nerve embedded in epoxy resin were used for imaging-based axon quantification in a masked fashion similar to our previous studies [25 (link)]. Images of the toluidine blue-stained sections were collected as non-overlapping tile images using Zeiss AxioObserver.Z1 microscope for wide-field fluorescence microscopy (Carl Zeiss, Thornwood, NY) and the Zen software (Blue Edition; Carl Zeiss) to allow axon counts representing the entire surface area of cross-sections as previously described [25 (link)]. Axon counting was performed by a researcher masked for the experimental group. After image acquisition, nerve outlines were manually traced on mosaics of images. Image processing determined the size and shape parameters to exclude intervening glia, myelin debris, and highly degenerated axons. The axon loss was determined by the ratio of axon counts in ocular hypertensive eye to normotensive fellow eye.

A CLSM study of whole skin and sectioned tissue was performed with an inverted Zeiss LSM 800 with Airyscan (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 10× objective lens. To visualize the fluorescence localizations of the rhodamine B base-loaded NBD-PE probed microemulsions on the skin surface, treated skin was placed onto a 22 mm × 50 mm coverslip (MENZEL-GLÄSER^®, Braunschweig, Germany) using a small volume of PBS as an immersion medium by orienting the epidermis toward the objective lens (EC Plan Neofluar 10×), which was equipped with four diode lasers. Green fluorescence (dye name AF488) was detected at excitation and emission wavelengths of 493 and 517 nm, respectively. Red fluorescence (dye name AF568) was detected at excitation and emission wavelengths of 577 and 603 nm, respectively. The sectioned tissue was observed using the same objective lens, for which the blue fluorescence from DAPI staining was detected at excitation and emission wavelengths of 353 and 465 nm, respectively. The fluorescence intensities of the image were analyzed by Zen software (Blue edition, Carl Zeiss Microscopy GmbH, Jena, Germany).

Epifluorescent images were collected by a fully automated slide scanner (AxioScan.Z1, Zeiss, Jena, Germany) equipped with an LED Light Source (Colibri 7, Zeiss, Jena, Germany). The appropriate excitation and emission filters (excitation/emission wavelengths in nm) were set as: 353/465 (DAPI), 488/509 (green), 548/561 (red), and 650/673 (infrared). A Plan-Apochromat 10 ×/0.45 objective for pre-focusing and a Plan-Apochromat 20 ×/0.8 objective for fine focus image acquisition was used. Offline image stitching (8 μm stacks, variance projection) for overviews of brain slices and further analysis was performed using ZEN software (Blue Edition, Zeiss, Oberkochen, Germany).
Confocal images were taken using a laser-scanning microscope (LSM-710, Zeiss) as previously described [23 (link)]. Figures presented in this work were modified with Zen 1 software (Black Edition, Zeiss, Oberkochen, Germany).

Samples were cytospun on microscopic slides (1000 rpm, 2 min) using StatSpin Cytofuge 2 (BeckmanCoulter, Marseille, France) and left to dry overnight. Slides were stained with May-Grünwald stain (50% working solution, 5 min) and, subsequently, Giemsa stain (10% working solution, 20 min). Morphology was examined using AxioVert 200 microscope and images were obtained using AxioCam MRc 5 camera and ZEN software, blue edition (Carl Zeiss AG, Oberkochen, Germany).

Wild type cells grown in YPD medium to late exponential phase were transformed with GFP-tagged PDE5 constructs and selected on YPD plated supplemented with 100 μg/mL G418. Transformed cells were stained with 100 nM MitoTracker (CMTMRos MitoTracker Orange, #M7510 Thermo Fisher Scientific Inc., Waltham, MA, USA), according to manufacturer’s instructions, to visualize mitochondrial compartment. Nuclei were stained with 2.5 μg/mL DAPI (Thermo Fisher). Cells from three independent experiments were visualized using a Zeiss LSM 800 System using the 488 (EGFP) and 576 nm (MitoTracker; Thermo Fisher Scientific Inc., Waltham, MA, USA) laser lines with corresponding band-path filters. Image recording was optimized using the Zeiss Zen Software Blue edition (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were acquired with a 20× PanFluor Objective (Carl Zeiss Microscopy GmbH, Jena, Germany) and imported into ImageJ (NIH, Bethesda, MD, USA), background was subtracted, and 16 fields (10 cells/field) for each condition were used to determine Pearson’s correlation coefficient (p) expressed as mean ± SEM. Statistical significance was calculated using the ANOVA one-way test with Holm–Sidak’s post hoc comparison. ** p = 0.007 (PDE5A2 vs. PDE5A1).