Dapi nuclear staining

Mice were sacrificed and perfused with 50 mL cold PBS, followed by 50 mL 4% paraformaldehyde. Cryosections were obtained of the coronal plain at 5 μm thickness. Non-specific staining was blocked by 1% BSA for 1 h, followed by incubation with primary antibodies against microglia surface marker CD11b (1:100 Cell Signaling, Danvers, MA, USA), M2 marker CD163 (1:120 Cell Signaling) for 2 h at 4 °C. Slides were washed thrice before staining with secondary antibodies: anti-rabbit (Cell Signaling) and anti-mouse (Cell Signaling). DAPI nuclear staining (Sigma-Aldrich) was added to the mounting solution. Primary-cultured microglia were stained as follows: cells were fixed with 4% paraformaldehyde for 1 h, washed thrice with cold PBS, and stained with antibodies against Iba-1(1:100, Cell Signaling) for 2 h with nuclear staining DAPI (Sigma-Aldrich) added at the last 15 min. Cells were washed twice before incubation with secondary antibodies: anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling). All images were taken by the Invitrogen EVOS FL Color imaging system.

Hearts were fixed in 10% formalin for 24 h, followed by an overnight dehydration in 70% ethanol. Hearts were sectioned into apex, mid and base, and processed for paraffin embedding. 10 µm thick slices of heart from the apex, mid and base regions were cut using a Reichert-Jung, 1140/autocut microtome and slide mounted for direct immunohistochemical fluorescent staining. Sections were deparaffinised with Histoclear, prior to rehydration and antigen retrieval (boiling in citrate buffer for 30 min). Immunofluorescent antibody labelling (c-kit, R&D; α-sarcomeric actin, Sigma; AlexaFluor-488 and -568 secondary antibodies, Thermo Fisher) proceeded according to standard methods, with DAPI nuclear staining (Sigma) and coverslip mounting with Slowfade (Life Technologies). For collagen staining, slides were rinsed with Histoclear twice, prior to rehydration and staining with Sirius Red for 60 min, followed by 0.01 M HCl wash to remove excess stain. The slides were dehydrated and mounted in DPX prior to viewing. Slides were viewed using a Zeiss LSM 700 confocal microscope, and image analysis (cell counts or area calculations) performed using ImageJ, with 18 random fields of view analysed per subject (six individual subjects per group).

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).