A11008

Primary antibodies used were: rabbit anti-dtTomato dsRed (1:1000; Clontech, 632496), anti-olig2 (1:500, Millipore, AB9610) and anti-cleaved caspase-3 (1:1000: Cell Signalling Technology, 9661); mouse anti-BrdU (IgG₁; 1:200, Sigma-Aldrich, B2531), anti-GFAP (IgG₁, 1:1000, Millipore, MAB360), anti-NeuN (IgG₁, 1:1500, Millipore, MAB377), anti-PCNA (IgG_2a; 1:150, Millipore, MAB424R) and anti-S100β (1:200, Abcam, Ab4066). Secondary antibodies of the relevant species and subclass, either coupled to biotin-Streptavidin Cy2 or Alexafluorochromes (-350, -488 or -568/594) were purchased from Thermo Fisher Scientific (A-11001, A-11004, A-11008 and A-11011) and Jackson ImmunoResearch (115-065-206 and 016-220-084), and used at previously described dilutions (Hajihosseini et al., 2008 (link)).

Primary antibodies are listed in Table 1. Secondary antibodies: anti-rabbit Alexa 488, anti-mouse Alexa 488, anti-rabbit Alexa 555, anti-mouse Alexa 555 from Life Technologies, (Carlsband, CA, USA), (1:500; A11008, A110296, A21428 and A21422) and anti-guinea pig Alexa 594 from Jackson ImmunoResearch (Cambridgeshire, UK), (1:500; 103-605-155).

Cross-sections (7 μm cryosections from formalin-fixed samples) were washed in PBS (3 × 5 min), permeabilized in 0.5% Triton X-100 (30 min), and blocked for non-specific binding in 5% goat serum containing 1% BSA (30 min). The sections were then incubated with primary antihuman polyclonal antibodies against Ki67 (rabbit IgG, 1:200, Thermoscientific, RB-1510-P0), CD45 (mouse IgG1, 1:1000, Abcam, ab33533), vimentin (mouse IgM, 1:2000, Abcam, ab20346), αSMA (rabbit IgG, 1:600, Abcam, ab5694), or collagen I (mouse IgG1, 1:200, Sigma, c2456), in 10× diluted block solution (overnight at 4 °C). After washing in PBS (3 × 5 min), the sections were incubated with 1:500 secondary goat antibodies labeled with Alexa-488 conjugate (antimouse IgG1 (for CD45, Molecular Probes, A21121) or antirabbit IgG (for αSMA, Molecular Probes, A11008)) or Alexa-647 conjugate (antimouse IgM (for vimentin, Jackson Immunoresearch, 115-605-075) or antimouse IgG1 (for collagen I and Ki67, Molecular Probes, A21240)), in 10× diluted block solution (60 min). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:500, Sigma). The stained sections were mounted in mowiol (Sigma, 81381) and visualized with an inverted epifluorescent microscope (Zeiss Axiovert 200M, ×20/0.5 Plan-Neofluar lens, three sections/sample).