D27802

For immunofluorescence, cells were fixed with 3.7% formaldehyde/1× PBS (Cat.No.: F8775, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 15 min and permeabilized with 0.7% Triton-X100 in 1× PBS for 20 min. All washing steps were performed with PBS-T (1× PBS/0.075% Tween-20). For detection of PCNA, cells were further incubated for 5 min in ice-cold methanol for antigen retrieval. Blocking (1% bovine serum albumin in 1× PBS) was performed for 30 min at room temperature. EdU was detected using the Click-IT assay as described by the manufacturer (1:1000 6-FAM azide or 1:2000 5/6-sulforhodamine azide; Cat.No.: 7806 and 7776, respectively, Carl Roth, Karlsruhe, Germany). Primary and secondary antibodies were diluted in the blocking buffer and incubated for 1 hr at room temperature with subsequent 3×10 min of PBS-T washing. DNA was counterstained with DAPI (4′,6-diamidino-2-phenylindole, 10 μg/ml, Cat.No.: D27802, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 10 min, and samples were mounted in Vectashield (Cat.No.: VEC-H-1000, Vector Laboratories, Inc, Burlingame, CA, USA). Antibody characteristics are summarized in Supplementary file 1d.

In order to test for the presence of additional axonal arborizations that might not have been resolved in the two-photon tomography, we collected the 50 μm-thick slices immediately after two-photon tomographical imaging. We then amplified the GFP signal with immunostaining. During this process, the slices were firstly incubated in blocking buffer 0.3% Triton (Applichem, Germany) and 2% normal goat serum (NGS, Vector, S-1000-L020) in PBS (0.9% NaCl, 0.01 M phosphate buffer, pH 7.4) for an hour. Then, we incubated the sample for 48 h shaking at 4°C in the primary anti-GFP antibody (rabbit polyclonal 1:5,000, Abcam 290, UK) together with 0.3% Triton X-100 in PBS, followed by two washes with PBS for 10 min. Subsequently, the slices were placed in the secondary antibody (goat anti-rabbit conjugated to Alexa 488 1:200, Life Technologies A-11012) together with 0.3% Triton X-100 in PBS for 2–2.5 h at room temperature. At the final step, the slices were washed in PBS three times for 10 min and mounted on Superfrost slides using, 4-Diazabicyclo[2.2.2]octane (DABCO, Sigma-Aldrich D27802, USA) as mounting medium. Images of the stained sections were obtained using the two-photon tomography microscope at the same laser power levels as the original imaging, enabling direct comparison.