Anti sox10

Cultured cells were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized with 0.2% Triton and 50 mM NH₄Cl. Fixed samples were pre-treated with BlockingOne (Nacalai Tesque) for 30 min at RT, and incubated with anti-OCT3/4, anti-NANOG, anti-SSEA4, anti-TRA1-60 (Cell Signaling Technology; diluted 1/200), anti-GFP (Molecular Probes; diluted 1/500), anti-SOX10 (SantaCruz Biotechnology; diluted 1/100) antibodies in 5% of BlockingOne in PBST for overnight at 4°C. After three washes with 0.1% Tween20 in PBS, cells were incubated with Alexa488 or Alexa594-conjugated secondary antibodies (Molecular Probes; diluted 1/500). Cells were washed and mounted in SlowFade Diamond antifade mountant with DAPI (Molecular Probes). Fluorescent images were collected on the software of BZ-X700 (Keyence). For quantitation of cultured cells, numbers of dishes were analyzed from triplicate experiments.

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 antibodies used for immunoblot analysis included IL-1β (1:1,000; R&D Systems), GROα (1:1,000; Thermo Fisher Scientific), IL-6 (1:1,000; R&D Systems), IL-8 (1:1,000; R&D Systems), IL-1R1 (1:1,000; R&D Systems), phosphorylated ERK (pERK; 1:5,000; Sigma-Aldrich), β-tubulin (1:5,000; Santa Cruz Biotechnology, Inc.), ERK2 (1:5,000; Santa Cruz Biotechnology, Inc.), pp65 (1:1,000; Cell Signaling Technology), p65 (1:1,000; Cell Signaling Technology), and BCL2 (1:1,000; Cell Signaling Technology). Anti–rabbit IgG-HRP (1:5,000) and anti–mouse IgG-HRP (1:5,000) were obtained from GE Healthcare, and anti–goat IgG-HRP (1:2,000) was obtained from Santa Cruz Biotechnology, Inc. The primary antibodies used for immunohistochemical analysis included anti–IL-1β (goat polyclonal; 1:50; R&D Systems), anti–IL-1R1 (goat polyclonal; 1:50; R&D Systems), anti-CD68 (clone KP1; mouse; 1:300; Dako), anti-CD163 (clone 10D6; mouse; 1:50; Thermo Fisher Scientific), anti-SOX10 (goat polyclonal; 1:120; Santa Cruz Biotechnology, Inc.), anti–IBA-1(rabbit polyclonal; 1:300; Wako Pure Chemical Industries), anti-SMA (clone 1A4; mouse; 1:200; Thermo Fisher Scientific), and anti-GROα (rabbit polyclonal; 1:50; Proteintech) followed by appropriate detection systems.

For Western Blot analysis, approximately 6x10⁵ cells were pelleted by centrifugation and lysed in 50μl of RIPA buffer (150 mM NaCl, 1% NP40, 0.5% EDTA, 0.1% SDS, 50 mM Tris pH 8, 0.5 mM protease inhibitor cocktail (Roche)) for 20 minutes at 4 °C with rotation. Samples were passed through a syringe needle, the supernatant was collected and protein concentration determined using a Bradford assay. 40 μg protein was loaded per well for SDS-PAGE using a Mini-PROTEAN Tetra cell system (BioRad) and Western blotting was performed with either anti-MITF antibody rabbit polyclonal (MERCK; HPA003259), anti-SOX-10 (Santa Cruz; sc-365692) or anti-β-ACTIN (Santa Cruz; sc-47778) mouse monoclonal antibodies. For cellular fractionation, approximately 1 x 10⁶ cells were harvested and pelleted at 4 °C. Cell pellet was re-suspended in 200 μl Lysis Buffer (15 mM HEPES, pH 7.5, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 250 mM Sucrose, 0.4% Igepal, 1 mM DTT, 0.5 mM, protease inhibitor cocktail (Roche), 100 U/ml RNAsin) and incubated at 4 °C for 20 minutes with rotation to lyse. Cells were further disrupted by pipetting and centrifuged at 2000 g for 10 minutes at 4 °C. Nuclear (pellet) and cytoplasmic (supernatant) fractions were collected. RNA was then prepared from each fraction and analysed by RT-qPCR.