Alexa conjugated fluorescent secondary antibodies

Paraffin sections were immunofluorescently labeled as described previously (26 (link)). Briefly, slides were deparaffinized, rehydrated and subjected to heat-induced epitope retrieval with Vector® Antigen Unmasking Solution (Vector). Sections were incubated overnight at 4°C with primary antibodies directed against GFP (Abcam), human collagen I (Abcam), and collagen I (Sigma Aldrich) and were visualized with Alexa-conjugated fluorescent secondary antibodies (Invitrogen) (Table 1). Sections were mounted with ProLong® Gold antifade reagent (Invitrogen) containing 4′,6-diamidino-2-phenylinodol (DAPI).

EPDCs cultured on UniFlex® Culture Plates were fixated in 4% paraformaldehyde (Merck) in PBS at RT for 15 min, permeabilized with 0.5% Triton X-100 (Merck) in PBS for 10 min and blocked with 5% BSA in PBS for 30 min. Subsequently, incubation with primary antibodies was performed overnight at 4°C in NET-gel (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.05% NP40, 0.25% gelatin) with 0.5% BSA. The antibodies and their suitable dilutions are listed in Table 1. Incubation for 2 h with Alexa-conjugated fluorescent secondary antibodies (Invitrogen) were used to visualize the primary antibody binding. Nuclear counterstain was achieved by DAPI (1:500) in NET-gel incubation for 5 min. Finally, cover glasses were mounted on microscopic slides with Mowiol.

Primary adult NSC neurosphere cultures were generated as previously described^{37 (link)}. Briefly, the lateral SVZs of five adult WT male mice were dissected per neurosphere culture and incubated in papain solution for 30 min at 37 °C with pipette dissociation every 10 min to obtain a single-cell suspension. Cells were then cultured in proliferating conditions (with addition of growth factors EFG and FGF) for 7 days at 37 °C to obtain primary neurospheres. To analyse cell differentiation, neurospheres were dissociated into single cells and plated on Poly-D-Lysine-coated glass coverslips in 24-well plates (50000 cells per well) in complete culture medium without proliferating factors for 1 to 5 days.
For immunocytochemistry experiments, cells were fixed with 4% PFA for 10 min at RT. Cells fixed on glass coverslips were blocked with 10% donkey serum and 1% BSA (Sigma) in PBS for 30 min at RT, and incubation with primary antibodies was performed in blocking solution overnight at 4 °C. After 3 × 5 min washes in 1X PBS at RT, cells were incubated with Alexa-conjugated fluorescent secondary antibodies (Invitrogen) (1/500, in 1% donkey serum and 1% BSA in PBS) for 1 h at RT. Cells were washed 3 × 5 min in 1X PBS, incubated with DAPI for 5 min at RT and mounted on SuperFrost+ glass slides (Thermo Fisher Scientific).