Axiocam mr r3

Bacterial strains grown in liquid culture were either directly applied to a microscope slide or previously immobilized on a 2% low-melting agarose in PBS agarose pad and air dried before microscopic analysis. Epifluorescence microscopy was performed using an Axio Imager.M2 light microscope (Carl Zeiss) equipped with Plan-Apochromat 63×/1.40 Oil M27 objective and the AxioCam MR R3 imaging device (Carl Zeiss). GFP, Alexa Fluor 488, eCFP and YFP fluorescence was visualized using filter set 38 (Carl Zeiss; excitation: 470/40 nm band pass (BP) filter; emission: 525/50 nm BP). Chlorophyll auto-fluorescence was recorded using filter set 15 (Carl Zeiss; excitation: 546/12 nm BP; emission: 590 nm long pass). When applicable, cells were previously incubated in the dark at RT for about 5 min with 10 µg ml⁻¹ DAPI in PBS to stain intracellular DNA. For visualization of DAPI fluorescence filter set 49 (Carl Zeiss; excitation: G 365 nm; emission: 455/50 nm) was employed. E. coli BL21 (DE3) cells expressing C-terminally GFP-tagged protein candidates were grown over night in LB and then diluted 1:40 in the same medium the following day. Cells were grown for 2 h at 37 °C, briefly acclimated to 20 °C for 10 min and induced with 0.05 mM IPTG at 20 °C. Protein localization of GFP/YFP-tagged proteins was then observed after indicated time points of cells immobilized on an agarose pad.

Live Z. mobilis cells were observed using a Zeiss Axio Imager Z2 microscope. The images were captured by Axiocam MR R3 (ZEISS) and analyzed by ZEN 2.3 pro software (ZEISS). For staining membrane, growing cultures were centrifuged (4500xg) for 5 min and the pellet was resuspended in phosphate buffered saline solution. FM4-64 (Thermo Fisher Scientific) was then added at a final concentration of (20 μg.ml⁻¹) and incubated for 15 min. Cells were then washed in PBS again and mounted on PBS agarose-pad (1% w/v). Comparison of cell size was done using the phase contrast images and the scale bars obtained from the imaging.

Sections were scanned for DiD dye positivity using Texas Red light with a confocal microscope (FV1000, Olympus), to locate the injury site, even after neuroregeneration was completed. For the quantification of IHC, Cresyl Violet, and SA β‐gal stainings, a Zeiss (“Axio Imager Z1”) fluorescence microscope equipped with a AxioCam MR R3 camera (fluorescence) and a Mrc5 color camera (Bright field) was used to photograph 3 sections per animal. 20X tile scans were stitched using ZEN software (ZEN Pro 2012, Carl Zeiss). For Picro sirius red staining, polarized light was used to visualize collagen fibers using a Leica DM6 microscope and LAS X software (Leica Microsystems). For greater detail, 63X with immersion oil was applied. Channels were equally intensified for all conditions using Adobe Photoshop SC5, figure configurations were made with Adobe Illustrator (Adobe Systems).

Live cell imaging was done using the Axio Observer.Z1 (Zeiss) microscope with alpha plan-apochromat 40x/Oil DIC (UV) M27 (Zeiss) in an environment chamber (5% CO₂; 37 °C) using Axiocam MR R3 (Zeiss). Cells were stained with MitoView red (Biotium) and the mitochondrial length and area were measured by using Image J software (detailed protocol in Supplemental Materials and Methods).

The proliferation of cells was detected via 5-Ethynyl-2′-deoxyuridine (EdU) incorporation according to the manufacturer’s instructions. Briefly, cells were incubated with 10 µM EdU in cell culture medium for 2 h under static or dynamic conditions. Cells were fixed with 4% PFA for 10 min, permeabilized with 0.5% Triton-X 100 for 20 min and afterwards incubated with the EdU-Click-ItTM reaction cocktail for 20 min. Nuclei were counterstained with 1 µg∙mL⁻¹ Hoechst 33342 in PBS for 15 min in the dark. Finally, cells were mounted and analyzed via confocal microscopy (LSM800, Carl Zeiss, Jena, Germany) or via epifluorescence microscopy (Axio Observer Z1, Carl Zeiss) equipped with a black and white camera (AxioCam MR R3, Carl Zeiss). All steps were conducted at room temperature.