Bovine serum albumin bsa

Cells were permeabilized for 30 min at room temperature with PBS 0.2% Bovine Serum Albumin (BSA, Euromedex, 04-100-812) 0.05% Saponin (Sigma-Aldrich, S4521). Cells were then incubated for 1 h at room temperature with primary antibody, followed by washing three times with PBS 0.2% BSA 0.05% Saponin and incubated protected from light for 20 min in the same buffer with spinned secondary antibodies. After washing once with PBS BSA Saponin, and once with PBS, coverslips were soaked three times in PBS, three times in water, and mounted on slides.
For regular confocal microscopy, coverslips were mounted with 4–6 µL of Fluoromount G (SouthernBiotech, 0100-01) on slides (KNITTEL Starfrost) and dried overnight protected from light before microscope acquisition.
For TIRF microscopy, following staining, with secondary antibody, coverslips were soaked in PBS and mounted with 4–6 µL of PBS, sealed with uncolored nail polish and dried for 15 min before acquisition.

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).