Alexa anti rabbit 488

For histopathology, samples were formalin-fixed, paraffin-embedded, and sectioned at 5 μm thickness. For each sample, a section was stained using a standard hematoxylin and eosin (H&E) protocol. For IHC of cultured spheres, samples were sectioned at 10 μm thickness after fixation with 2% PFA in PBS for 2 hours on ice, and cryo-protected with 30% sucrose in PBS, followed by staining without heat antigen retrieval. Primary antibodies are listed in Table S2. Fluorescently coupled secondary antibodies, including Alexa anti-rabbit 488, anti-mouse 488, and anti-rabbit 647 (Invitrogen, Grand Island, NY, USA) were used. Immunohistochemistry to detect cilia was performed on Myc, MycVD and Myc/ΔPOZ tumor sections and tumorspheres with antibodies to ARL13B and γ-tubulin (See Table S2) and with DAPI. Representative images of each sample/stain combination were captured and analyzed using Axiovision software (Carl Zeiss Microscopy, Thornwood, NY, USA). Immunoblotting was performed on tumor cells purified from mouse G3 MB (Myc), MycVD and Myc/ΔPOZ tumor, lysed in RIPA buffer and proteins separated on SDS/PAGE gels detected with primary antibodies against Myc, β-actin, and GAPDH (see Table S2), as previously described (Ayrault et al., 2010 (link); Kawauchi et al., 2012 (link)).

Third instar larvae were dissected, fixed, and immunostained as described (Fye et al 2010 , Gunawardena & Goldstein 2001 (link)). Briefly, larvae were dissected in dissection buffer (2X stock contains 128 mM NaCl, 4 mM MgCl₂, 2 mM KCl, 5 mM HEPES, and 36 mM sucrose, pH 7.2). Following dissection, larvae were treated with 0, 10, or 50 mM EtOH at 25°C for 20 min. Larvae were fixed in 4% formaldehyde and incubated with primary antibodies against either rabbit monoclonal cysteine string protein (DCSP-3, 1:10, Developmental Studies Hybridoma Bank; RRID:AB_528184) overnight. Larvae were incubated with the neuronal membrane marker Texas Red-conjugated horse radish peroxidase (HRP) and secondary antibody (Alexa anti-rabbit 488, 1:100, Invitrogen) for 2 hrs at room temperature, mounted using Vectashield mounting medium (Vector Labs) and imaged using a Nikon TE-2000E inverted microscope at 60X.