Role of Shh in Disc Maintenance Figure 10. Shh is the key regulator of other major cell signaling pathways. Figure 10 shows that cyclopamine treatment in vitro, and targeting of Shh in vivo both cause alterations in activities of other signaling pathways. (A) and (B) show increases in BMP signaling and (C) shows the graph for the intensity of immunostaining from both the experiments. (D) and (E) show decreases in TGFb signaling represented by graph plotted for the intensity of immunostanining (E). (F) shows an increase in canonical Wnt signaling. In (A)–(D), red = expression of the specific protein. In E, blue = b-gal positive cells, pink = nuclei stained with nuclear fast red. doi:10.1371/journal.pone.0035944.g010 Immunolocalization analysis Immunostaining was carried out on 6 mm thick coronal sections of IVDs from in vitro and in vivo experiments. The sections were fixed in 2% PFA for 2 minutes, permeabilized using 1x PBS containing 0.2% Triton-x100 for 20 minutes, blocked in blocking buffer (10% donkey or goat serum, 4% bovine serum albumin in 1x PBS containing 0.1% Triton-x100 [PBST]) for one hour, treated with diluted primary antibody (Brachyury, sc-17743, Santa Cruz; Sox9, Ab3697, Abcam; Cytokeratin 19, TROMA-III, Hybridoma Bank; Collagen I, GTX41285, Gene TexH, Inc; Collagen II, NB600-844, Novus Biologicals; Chondroitin sulfate, ab11570, Abcam; Keratan sulfate, 270427-1, Associates of Cape Cod Inc.; Aggrecan, ab3778, Abcam; Shh, S4944, Sigma-Aldrich; Ptch1, MAB41051, R? Gli1, AF3455, R? Ihh, ab52919 (also PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e35944