Fp1012

The decalcified mandibles were dehydrated in serial sucrose/PBS solutions and embedded in OCT compound (Tissue-Tek, Sakura). OCT-embedded samples were cryosectioned at 8 μm using a cryostat (Leica CM1850) followed by staining. For immunofluorescence staining, cryosections were soaked in blocking solution (PerkinElmer, FP1012) for one hour at room temperature and then incubated with primary antibodies diluted in blocking solution at 4°C overnight. After washing three times in PBS, the sections were incubated with alexa-conjugated secondary antibody (Thermo Fisher) and counterstained with DAPI (Thermo Fisher Scientific, 62248). The primary antibodies are listed in Table S1. For in situ hybridization analysis, cryosections were stained with RNAscope Multiplex Fluorescent kit (Advanced Cell Diagnostics, 323100) or RNAscope 2.5 HD Reagent Kit-RED assay (Advanced Cell Diagnostics, 322350) according to the manufacturer’s instructions. All of the probes are listed in the Key resources table.

To address the PTEN deletion following AAV-Cre injection, free-flating coronal sections through the brain were incubated in 1% hydrogen peroxide for 15 min. After blocking in Tyramide Signal Amplification (TSA) blocking buffer (0.5g blocking reagent/ 40ml TBS, PerkinElmer, FP1012) sections were incubated in primary antibody at RT overnight (rabbit anti-PTEN, Cell Signaling 9188S, 1:250). Sections were washed in TBS and incubated with secondary antibody (donkey anti-rabbit HRP, Jackson Immunolabs 711-065-152, 1:250) in TSA blocking buffer for 2 hours at RT. Following a wash in TBS, sections were stained with 3–3′ diaminobenzidine (DAB, Vector Labs SK-4100) for 5 min, rinsed in TBS, mounted on gelatin subbed slides in weak mounting solution (0.5% gelatin and 0.05% chromium potassium sulfate, Sigma Aldrich, St. Louis, MO), air-dried and cover-slipped with DPX mounting medium.

Sections were immersed in a preheated antigen unmasking solution (Vector, H-3300) in a microwave oven at 95°C for 15–20 minutes, followed by cooling at room temperature for 20 minutes and incubation with a blocking reagent (PerkinElmer, FP1012) for 1 hour and then a primary antibody overnight at 4°C. The following primary antibodies were used in our study: active Caspase-3 (Casp3; Abcam, ab2302; 1:100), phospho-Histone H3 (pH3; Millipore, 06–570; 1:100), non-muscle myosin heavy chain IIA (NMHCIIA; Biolegend, 909801; 1:500), phospho-Smad2 (pSmad2; Cell Signaling, 3108; 1:500), phospho-Smad1/5/9 (pSmad1/5/9; Cell Signaling, 13820; 1:500), ΔNp63 (p63; Biolegend, 619002; 1:100), Sox2 (Abcam, ab97959; 1:1000), Sox2 (Santa Cruz, sc-17320; 1:100), and SSEA1 (Developmental Studies Hybridoma Bank, AB 528475; 1:10). After three washes in PBS, sections were incubated with the Alexa Fluor 568 or Alexa Fluor 488 (1:200, Invitrogen) secondary antibody for 2 hours at room temperature. For pSmad2 and pSmad1/5/9, HRP-labeled goat anti-rabbit IgG (PerkinElmer, NEF812001EA; 1:200) was used as secondary antibody and a TSA kit was used for signal detection (PerkinElmer, NEL741001KT). Sections were counterstained with DAPI (Sigma, D9542). Images were captured using a fluorescence microscope (Leica DMI 3000B) with filter settings for DAPI/FITC/TRITC.

For immunofluorescent staining, sections were rinsed 3 times for 2 min in 0.01M PBS containing 0.2% Triton X-100 (Tx) (Sigma-Aldrich, T8787) and then pre-treated with 3% H2O2 in PBS-Tx for 10 min. Sections were rinsed 3 times for 2 min in PBS-Tx, and incubated in tyramide signal amplification (TSA) blocking reagent (PerkinElmer, FP1012) for 1 hr. Then the sections were incubated with primary antibody solutions containing rabbit anti-GFAP (z0334; DAKO) and mouse anti CD11b/c (Abcam, ab1211) in TSA blocking reagent in PBS-Tx for 24 hr at 4 °C. After primary incubation, the sections were rinsed 3 times for 2 min in PBS-Tx, and then incubated them for 2 hr in the secondary antibody solution containing goat anti-rabbit Alexa Fluor 488 [1:300] (Life Technologies, A11034) and goat anti mouse Alexa Fluor 647 [1:300] (Life Technologies, A21236) in TSA blocking reagent in PBS-Tx. The sections were rinsed 3 times for 2 min in 0.1M PBS, then were mounted onto glass slides and coverslipped with ProLong Antifade Reagent with DAPI nuclear stain (Life Technologies, P36931).