Smoothened

Cells grown on cover slips were fixed with 3% formaldehyde for 7 min and then with ice cold 70% MeOH for 1 min and permeabilized in 0.5% Triton X-100. For BrdU staining, DNA was denatured in 2 N HCl for 30 min. After antibody treatment and staining with 4,6-diamidino-2-phenylindole, cover slips were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Samples were incubated with primary antibodies for BrdU (BU20A), CenPF, PCNT, phospho-H3 (Santa Cruz, Santa Cruz, CA, USA), γ-tubulin, acetylated tubulin (Sigma, St Louis, MO, USA), phospho-Rb (Cell Signaling, Beverley, MA, USA) and Smoothened (Abcam, Cambridge, UK). Secondary antibodies were from Sigma.
Fibroblasts were grown to 70–80% confluency followed by serum starvation in MEM containing 0.1% fetal calf serum (FCS) for 2–3 days to promote entry into G0. Cells were processed for IF as mentioned earlier and cilia visualized with anti-acetylated tubulin and γ-tubulin antibodies.

For histological analysis, animals were euthanized at specified time periods and tissues were fixed in 4% paraformaldehyde. Histological sectioning, hematoxylin-eosin (H&E) staining and Masson Trichome staining were performed as previously described^{5 (link)}. Immunohistochemistry was performed on cryosections (10 µm thick) using standard procedures^{5 (link),50 (link)–52 (link)}. Briefly, sections were rehydrated, permeabilized, and blocked with 10% normal donkey serum (NDS), 0.1% Triton X-100 in PBS at room temperature and incubated overnight at 4 °C with primary antibodies: α-actinin (Abcam; 1:300), desmin (Novus biologicals; 1:300), Shh (Santa Cruz Biotechnology; 1:200), cleaved caspse-3 (Cell Signaling; 1:300), endomucin (Abcam; 1:100), SM22 (Abcam; 1:400), α-phospho-Histone H3 (Ser10) (Millipore; 1:100), Ki67 (Abcam; 1:200), PCNA (Santa Cruz Biotechnology; 1:100), Mef2a (Santa Cruz Biotechnology; 1:100), Smoothened (Abcam; 1:200), Nkx 2–5 (Santa Cruz Biotechnology; 1:100), and GFP (ThermoFisher Scientific; 1:300) sera. Sections were rinsed and incubated with combinations of secondary antibodies (1:400) including Alexa 488, Alexa 594, Cy3, and Cy5 (Jackson ImmunoResearch Laboratories). EdU staining was performed using the EdU labeling kit (Life Technologies).

The rats were euthanized and decapitated, and their ischemic ipsilateral cortices were rapidly dissected on ice and stored at −80 °C until needed. The samples were then taken from the freezer and sonicated in RIPA buffer (Beyotime Institute of Biotechnology, China) containing phosphatase and protease inhibitor cocktails. A BCA Kit (Beyotime Biotechnology) was then used to determine the protein concentrations. Equal amounts of sample were separated by SDS-PAGE and transferred onto PVDF membranes, which were blocked in 5% non-fat dry milk at room temperature for 1 h. The membranes were then incubated overnight at 4 °C with primary antibodies against VEGF (1:1000 Bioworld Technology, USA), Ang-1 (1:1000 Bioworld Technology, USA), Shh (1:250 Life Technology, USA), Patched-1 (1:1000 Abcam, UK), Smoothened (1:1000 Abcam, UK) and Beta-actin (1:5000 Bioworld, USA). The next day, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized with chemiluminescence reagents that were provided with an ECL kit (Bioworld, USA). Blot intensity was detected using Image J software.