Reflected fluorescence microscopy system

To assess the amount of TH-positive (TH +) cells in six nigral serial sections per animal, the stereological optical fractionator method was used [54 (link), 55 (link)]. Cells were stereologically extrapolated using the “Optical Fractionator Probe” of Stereo Investigator 10.40 software (MicroBrightField, Stereo Investigator, RRID:SCR_018948) with a grid size of 200 × 200 µm and a counting frame of 100 × 100 µm. The analysis was performed using a × 40 objective of a BX53 microscope (Olympus) attached to a DV‐47d camera (MicroBrightField). Results are expressed as the ratio of contralateral to ipsilateral cell numbers.
Striatal and nigral photos were captured using a light microscope (Zeiss Axio, Carl Zeiss AG) connected to ProgRes® CapturePro 2.8.8 (Jenoptik AG). Images were converted using ImageJ 1.52a (Wayne Rasband, ImageJ, RRID: SCR_003070).
Fluorescent transplant photos were visualized with an Olympus CKX41 microscope combined with a reflected fluorescence microscopy system (Olympus Corporation). Photos were captured using a ProgRes® CapturePro 2.8.8 camera combined with the ProgRes CapturePro 2.10 software (both Jenoptik AG), and overlays were merged by the use of ImageJ 1.52a (Wayne Rasband, ImageJ, RRID: SCR_003070).

Whole constructs were examined for viability using propidium iodide/fluorescein diacetate staining (PI/FDA; Sigma-Aldrich, Saint Louis, MO, USA) on days 1, 7, and 28 of osteogenic maintenance. After washing with PBS (Merck, Berlin, Germany), staining was performed, first in an FDA solution (3 μg mL⁻¹; 15 min, 37 °C) and then in a PI staining solution (100 μg mL⁻¹; 2 min; room temperature). For microscopy, an Olympus CKX41 combined with a reflected fluorescence microscopy system was used (Olympus, Hamburg, Germany). The staining results were photodocumented using the ProgRes^® speed XT core 5 camera and ProgRes^® CapturePro 2.10 software (both Jenoptik, Jena, Germany).