Hoechst 33258 nuclear staining

After 48 h, brightfield images with a phase contrast of DRG-explants were taken at 5× magnification using a Zeiss Axio Vert.5A1 inverted fluorescent microscope (Carl Zeiss AG, Oberkochen, Germany) and a Zeiss AxioCam MRc camera. DRG explants were then fixed in 4% PFA at RT for 10 min and permeabilized and blocked in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) (i.e., the dilution buffer) for 20 min at RT. For immunocytochemistry, the cultures were incubated overnight at 4 °C with the primary antibody monoclonal mouse anti-β-Tubulin III (1:1000, Sigma-Aldrich, Buchs, Switzerland, Cat. No. T8578) for regenerating axons. The cultures were then washed in PBS and incubated for 1 h at RT with the secondary antibody sheep anti-mouse Cy3 (1:500, Sigma Aldrich, Cat. No. C2181) and Hoechst 33258 nuclear staining (1:1000, Sigma Aldrich, Cat. No. 94403). Digital images of the stained specimens were acquired at 15× magnification (numerical aperture 0.45) using a Nikon Eclipse Ti₂ inverted fluorescent microscope and a Photometrics prime 95B 25 mm camera (Teledyne Photometrics, Tucson, AZ, USA). The images were automatically stitched by the Nikon NIS-Elements AR image analysis software.

After 48 h, DRG cultures were observed under a microscope and bright-field images with phase-contrast were taken at 5× magnification using a Zeiss Axio Vert.A1 inverted fluorescent microscope (Carl Zeiss AG, Jena, Germany) and a Zeiss AxioCam MRc camera (Carl Zeiss AG, Jena, Germany). DRG-explants were then fixed in 4% PFA at room temperature (RT) for 10 min and permeabilized and blocked in PBS containing 0.1% Triton X-100 and 1% BSA (i.e., dilution buffer) for 60 min at RT. For immunocytochemistry, the cultures were incubated overnight with the following primary antibody at 4 °C: monoclonal mouse anti-β-Tubulin III (1:1000, Sigma-Aldrich, Cat. No. T8578) for axons. The cultures were then washed in PBS and incubated with the following secondary antibody: sheep anti-mouse Cy3 (1:500, Sigma Aldrich, Cat. No. C2181) and Hoechst 33258 nuclear staining (1:1000, Sigma Aldrich, Cat. No. 94403) for 60 min at RT. Subsequently, digital images were acquired at 10× magnification (numerical aperture 0.45) by using a Nikon Eclipse Ti inverted fluorescent microscope and a Photometrics prime 95B 25 mm camera.