Sc 365692

Cells were washed twice with PBS containing CaCl₂ and then lysed in a 100 mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris pH 8.0 RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitors (Sigma‐Aldrich). Protein expression was examined by Western blot using the anti‐ZEB1 (1/200, Sigma HPA027524, RRID:AB_1844977), anti‐ZEB2 (1/500, Sigma HPA003456, RRID:AB_10603840), anti‐MITF (clone C5, ab80651, 1/500, Abcam, RRID:AB_1603129), anti‐SOX10 (Santa Cruz, sc‐365692, RRID:AB_10844002) antibodies for primary detection. Loading was controlled using the anti‐GAPDH (1/20,000, Millipore) antibody. Horseradish peroxidase‐conjugated rabbit anti‐mouse, goat anti‐rabbit, and donkey anti‐goat polyclonal antibodies (Dako, Glostrup, Denmark) were used as secondary antibodies. Western blot detections were conducted using the Luminol reagent (Santa Cruz).

Tissue was embedded in paraffin as previously described (May et al., 2015 (link)). Immunofluorescence was performed either on paraffin-embedded tissue or on whole-mount dissected embryonic salivary glands and explant cultures (Gaete et al., 2015 (link)). Primary antibodies and dilutions were used as follows: anti-Sox9 1:300 (AB5535, Millipore); anti-BrdU 1:500 (ab6326, Abcam), anti-Sox2 1:200 (#2748, Cell Signaling Technology); anti-Mist1 1:50 (sc-98771, Santa Cruz Biotechnology), for which signal was amplified with the TSA kit (PerkinElmer); anti-laminin 1:300 (L9393, Sigma); anti-K5 1:300 (119-13621, Cambridge Bioscience); anti-Sox10 1:100 (sc-365692, Santa Cruz Biotechnology) using the TSA kit; anti-cleaved caspase 3 1:200 (#9661, Cell Signaling Technology). In situ hybridisation was performed as previously described (Gaete et al., 2015 (link)). Plasmids for probe generation have been described previously: Spry1 (Minowada et al., 1999 (link)), Fgf10 (Bellusci et al., 1997 (link)), Myb (Matalová et al., 2011 (link)), Etv5 (Hippenmeyer et al., 2002 (link)) and Col2a1 (Ng et al., 1997 (link)).

The tissue sections were processed using the TSA kit (Perkin Elmer) according to the manufacturer’s instructions. The deparaffinization of fixed tissues were performed by immersing the slides in tissue clear and rehydrated with graded ethanol (100–70%) and water. The heat-induced antigen retrieval was performed using citrate buffer (1.8 mM citric acid, 8.2mM sodium citrate, pH 5.0). The slides were treated with 3% H₂O₂-MetOH for 10 min to inactivate endogenous peroxidase activity. The tissues were blocked for 30 min with TNB buffer (0.1M Tris–HCL (pH 7.5), 0.15M NaCl, 0.5% blocking reagent from TSA kit). The primary antibodies were incubated overnight at 4 °C and biotinylated secondary antibody for 60 min at room temperature. The primary antibodies include polyclonal rabbit IGSF3 (1:750 HPA036305, Sigma-Aldrich), polyclonal mouse SOX10 (1:500 SC-365692, Santa Cruz). The reddish brown color was the stain generated by secondary biotinylated antibodies which includes goat-anti rabbit IgG (E0432, Dako) and goat-anti mouse IgG (E0433, Dako). The washes were performed with TNT (Tris/NaCl/Tween 20) buffer. The Mayer’s hematoxylin (S3309, Dako) was used to counterstain the tissue sections. For tissue morphological assessment, hematoxylin and eosin staining was performed. Whole stained slides were digitally scanned using a slide scanner (3Dhistec).