2.7. Microscopic imaging

Sections were analyzed using a ZEISS Axio Imager. M2 microscope, equipped with ApoTome.2 and a Camera Axiocam 702 mono (Zeiss, Germany). Specific filter cubes were used for the visualization of green (Filter set 38 HE eGFP shift-free (E) EX BP 470/40, BS FT 495, EM BP 525/50), red (Filter set 43 HE Cy 3 shift-free (E) EX BP 550/25, BS FT 570, EM BP 605/70), far-red (Filter set 50 Cy 5 shift-free (E) EX BP 640/30, BS FT 660, EM BP 690/50), and blue (Filter set 49 DAPI shift-free (E) EX G 365, BS FT 395, EM BP 445/50) fluorescence. Different magnifications were selected using a Zeiss x20 objective (Objective Plan-Apochromat 20x/0.8 M27 (FWD = 0.55 mm)) and a 63 × oil-immersion objective (Objective C Plan-Apochromat 63x/1.4 Oil DIC M27 (FWD = 0.14 mm)). To create photomontages, images were acquired using ZEN 2.3 pro software using Z-Stack and Tiles/Positions ZEN modules for each fluorophore, sequentially. Quintuple-ApoTome frames were collected stepwise over a defined z-focus range corresponding to all visible fluorescence within the section: multiple-plane frames were collected at a step of 1 μm while using x20 objective (between 4 and 10 frames per image). All images were then saved in. cvi, processed to get orthogonal and maximal intensity projections, and finally exported in. tiff. For the processing steps (i.e., adjust brightness and contrast, change colors, and merge images using Adobe Photoshop (Adobe Systems, San Jose, CA)).