Ab 59705

Tooth cultures were fixed overnight at 4°C in 4% paraformaldehyde (SigmaAldrich). Subsequently, they were cryoprotected overnight at 4°C in 30% sucrose and then embedded in OCT (Tissue-Tek) and stored at −80°C until use. Frozen sections (15 μm) were obtained using a cryostat (Shandon) and post-fixed in 4% paraformaldehyde for 10 min. Some sections were stained with hematoxylin-eosin or permeabilized using 1% Triton X-100 (SigmaAldrich) in PBS (SigmaAldrich) and blocked with 10% fetal bovine serum for 1 h at room temperature. These samples were incubated overnight at 4°C with the following antibodies:mouse anti-bromodeoxyuridine-fluorescein monoclonal antibody 1:200 (Roche 11202693001), rabbit anti-cleaved Caspase3 1:200 (Asp175; Cell Signaling) anti-Nestin 1:100 (ab5968; Abcam) and anti-Amelogenin 1:200 (ab59705 Abcam) antibodies. The secondary antibody used was Alexa Fluor 488-conjugated anti-rabbit antiserum (dilution 1:500). Cell nuclei were stained with DAPI (4′,6-Diamidino-2-phenylindole, SigmaAldrich) 0.5 μg/ml. Immunofluorescences were performed by triplicate and images were obtained using an Olympus FluoView FV500 confocal microscope or a Zeiss Axioskop fluorescence microscope with Nikon DS-Qi1Mc and Nikon NIS-Elements software, respectively.

After deparaffinization and rehydration, mandible tissue sections from 1 week old WT and AMELX KO mice were permeabilized for 10 min in 1% Triton X-100 (Thermo Fisher Scientific Inc, Waltham, MA, USA), then rinsed with 1× DPBS 3 times for 5 min each under agitation. Nonspecific binding sites were blocked by 30 min incubation in 1% bovine serum albumin (BSA, Euromedex, Mundolsheim, France) diluted in DPBS. Sections were incubated overnight at 4°C with primary anti-AMBN antibody (1/200) (sc 50534 (M-300), Santa Cruz Biotechnology) and anti-AMELX antibody (1/200) (ab 59705, Abcam). After washes with 1× DPBS, Alexa Fluor 594 or 488 Goat Anti-Rabbit IgG antibodies (1/500) (Thermo Fisher Scientific Inc) were applied for 1 h at room temperature followed by DAPI nuclear staining (1/100,000) (Sigma-Aldrich Co.). Sections were mounted using an aqueous mounting medium (Fluoprep, Biomérieux, France).

After deparaffinization and rehydration, endogenous peroxidase was inactivated by treatment with 3% H₂O₂ (Merck, Darmstadt, Germany) in 1× DPBS for 15 min. Sections from 1 and 8 week old WT and AMELX KO mice were then rinsed in 1× DPBS and blocked overnight at 4°C with ready-to-use (2.5%) normal horse blocking reagent (ImmPRESS reagent kit, Vector Laboratories, Burlingame, CA, USA). Sections were then probed with primary anti-AMBN antibody (1/100) (sc 50534 (M-300), Santa Cruz Biotechnology - rabbit polyclonal IgG to AMBN - Immunogen: amino acids 108–407 mapping at the C-terminus of AMBN mouse origin - Reacts with mouse) and anti-AMELX antibody (1/100) (ab 59705, Abcam, Cambridge, MA, USA - rabbit polyclonal IgG to AMELX - Immunogen: purified full length native protein of AMELX cow origin – Reacts with mouse, rat and cow) for 1 h at room temperature. Tissue sections were washed three times with 1× DPBS for 5 min and incubated for 30 min with ImmPRESS reagent (ImmPRESS reagent anti-rabbit Ig Kit, Vector Laboratories, Burlingame, CA, USA). After three washes in DPBS 1X, immuno cross-reactivity was visualized using diaminobenzidine peroxidase substrate (Novared, Vector Laboratories). Sections were dehydrated and mounted in resin (Eukit, Agar Scientific, Freiburg, Germany). Sections with no primary antibodies were used as negative controls.