Alexa fluor 546 conjugated goat anti mouse

Microglomeruli were labeled and quantified adapting a published protocol for double staining pre-synaptic and post-synaptic profiles (Groh et al., 2006 (link); Krofczik et al., 2008 (link); Hourcade et al., 2010 (link)). Brains were embedded in 5% low melting point agarose (Agarose II, no. 210–815, AMRESCO) and sectioned in a frontal plane (200 μm) with a vibrating microtome (Leica VT 1000S). Free-floating sections were repeatedly washed (3 times for 10 min) in PBS with 2% Triton X-100 and pre-incubated in PBS with 0.2% Triton X-100 and 2% normal goat serum for 1 h at RT. Preparations were then incubated for 4 days at 4^oC simultaneously in 0.2 U of Alexa Fluor 488 phalloidin (Invitrogen, A-12379) and a monoclonal anti-synapsin I antibody (1:50; SYNORF1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). After repeated washes (5 times for 10 min) in PBS, preparations were incubated in the secondary antibody (Alexa Fluor 546-conjugated goat anti-mouse, Invitrogen: 1:250 in PBS with 1% normal goat serum) for 2 h at RT. Brains sections were washed (5 times for 10 min) in PBS, transferred to 50% glycerol in PBS for 15 min and mounted on coverslips with 80% glycerol/PBS solution.

HEK293 cells plated on coverslips were transfected with Flag-DCC plasmids using the calcium phosphate method as described previously [55 (link)]. Approximately 40 h after transfection, the cells were incubated with 100 μg/mL myc-Netrin-1 or myc-Netrin-1 (∆407–443) for 30 min before being washed for 5 min with HBHA (20 mM HEPES, pH 7.0, and 0.5 mg/mL BSA in HBSS), rinsed with 1× PBS, and fixed sequentially with 4% paraformaldehyde in PBS for 10 min. The primary antibodies that were used were rabbit anti-myc (1:200, Abcam, ab9106, Cambridge, MA, USA) and mouse anti-flag (1:100, Sigma Aldrich, F3165) antibodies. The secondary antibodies that were used were Alexa Fluor 488-conjugated donkey anti-rabbit (1:1000; Invitrogen, A21206) and Alexa Fluor 546-conjugated goat anti-mouse (1:1000; Invitrogen, A10036) antibodies. The nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI) (Thermo Fisher, Waltham, MA, USA). All images were acquired with a Zeiss LSM 880 confocal microscope.

Similarly as described, the mandible tissues were collected and served for preparing serial sections, followed by Hemotoxylin and Eosin (H&E) staining, Masson–Goldner staining (Sigma-Aldrich, Burlington, VT, USA) (Fig. 7), or immunofluorescent staining (Figs. 8 and 9).
To evaluated osteogenic and angiogenic markers, the sections were immunostained with the primary antibodies: anti-CD31 (1:100, rabbit monoclonal, Abcam, Cambridge, UK), anti-ALP (Santa Cruz Bio. Inc., CA, USA), and anti-OCN (Santa Cruz Bio. Inc., CA, USA). For secondary antibodies, Alexa Fluor 488-conjugated goat-anti-rabbit, or Alexa Fluor 546-conjugated goat-anti-mouse (Invitrogen, Carlsbad, CA, USA) was used. After nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA), sectional images were obtained using an inverted fluorescence microscope (BZ9000). To evaluate the fluorescent signals of CD31, ALP, or OCN, three ROIs were set on each 20 × picture to include bone defect and the newly regenerated bone. The signals were measured three times to three times to be averaged using an imaging software, Image J (National Institutes of Health, USA). The co-stained DAPI signals were used for normalization of the cell number in each ROI, to calculate the percentage of positive area (%). The data in each group was expressed as mean ± S.E.M (Number of rats = 3 per group).