Gelatin block

Back skin or oral tissues (tongue) from p45 animals were fixed in 4% Paraformaldehyde for 0.5–1 hr followed by PBS washes. Tissues were incubated in 15% and 30% sucrose gradient at 4°C prior to embedding in O.C.T compound (Tissue Tek, cat. #: 4583) without prior fixation. Tissues were sectioned to 8µm on a Leica CM1950 cryostat using DB80LX blades (cat. #: 14035843496), mounted on SuperFrost Plus glass slides (Fisher Scientific, cat. #: 12–550-15), and dried 10 min at 37°C. Fresh frozen tissue from p0 mice and fixed p45 skin sections were fixed for 5 min with 4% PFA, washed with PBS, and blocked in a gelatin block (5% normal donkey serum (Jackson Immunoresearch, cat. #: 017–000-121), 3% BSA, 8% gelatin (Sigma cat. #: G7765), and 0.05% Triton X-100 in PBS) for 1 hr. Sections were stained with primary antibodies diluted in gelatin block and incubated overnight at 4°C, washed three times with PBS, incubated 2 hr with secondary antibodies at room temperature, counterstained with DAPI for 5 min at room temperature, and mounted in ProLong Gold (Invitrogen, cat. #: P36930). Tissues were stained with primary and secondary antibodies (Table S6) and images acquired using LAS AF software on a Leica TCS SPE-II 4 laser confocal system on a DM5500 microscope with ACS Apochromat 20 3 /0.60 multi-immersion, ACS Apochromat 40 3 /1.15 oil, and ACS Apochromat 63 3 /1.30 oil objectives.