Anti acetylated alpha tubulin

We grew RMS and EWS cells in 8-well chamber slides (Nunc, Rochester, NY). Cells were fixed with 4% paraformaldehyde/0.5% TritonX-100 in PBS for 30 min at room temperature with rocking. Cells were washed again with PBS at room temperature and were then blocked for 1 h with 10% donkey serum. We incubated the cells with anti-acetylated alpha-tubulin (Sigma, St. Louis, MO, 1:5000 dilution), and pericentrin (Abcam, Cambridge, MA, 1:500 dilution) or pericentrin2 (Santa Cruz Biotech, Santa Cruz, CA, 1:500 dilution) antibodies overnight at 4 °C. Cells were washed with PBS and incubated with the secondary antibodies at 1:300 dilution (donkey anti-goat IgG Alexa488, [Alexa, Eugene, OR], or donkey anti-mouse IgG Alexa568, [Alexa, Eugene, OR]) for 1 h at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenyindole (DAPI) at 1:2000 dilution (Biotium, Hayward, CA) for 20 min at room temperature with rocking. Immunofluorescence was observed using a Zeiss LSM510 or LSM880 META confocal laser scanning microscope.

Immunohistochemical staining or IHC paired with FISH was performed as described in Perillo et al., 2021 (link). Briefly three dpf plutei were fixed in 4 % paraformaldehyde (PFA) in filtered sea water (FSW) for 15 min at room temperature (RT). FSW was removed and samples were incubated in 100 % methanol for 1 min at RT, washed multiple times with phosphate buffer saline with 0.1 % Tween 20 (PBST) and incubated blocking solution containing 1 mg/ml Bovine Serum Albumin (BSA) and 4 % sheep in PBST for 1 hr. Primary antibodies were added in the appropriate dilution and incubated for 1 hr and 30 min at 37 °C. Anti-acetylated alpha tubulin (Sigma-Aldrich T67930) was used to label cilia and microtubules (1:200), Anti-Msp130 (gift from Dr. David R. McClay) to label skeletogenic cells (undiluted), 1E11 (gift from Dr. Robert Burke) to mark the nervous system (1:20), 5c7 (gift from Dr. David R. McClay) to label the endoderm (undiluted), Sp-An to label the post-oral and lateral neurons (1:250), Sp-Th (Sigma-Aldrich AB152) to label catecholaminergic neurons (1:100) and Sp-Chat (GeneTex GXGTX113164S) to label cholinergic neurons (1:100). Specimens were washed multiple times with PBST and incubated for 1 hr with the appropriate secondary antibody (AlexaFluor) diluted 1:1000 in PBST. Larvae were washed several times with PBST and imaged using a Zeiss LSM 700 confocal microscope.

Immunohistochemistry of tissue sections was performed as previously described (Satoh et al. 2007) using anti‐acetylated alpha‐tubulin (Sigma, 1/200), anti‐fibronectin (Sigma, 1/100), anti‐type II collagen (DSHB, 1/100), anti‐MHC (DSHB, Iowa City, IA, 1/50), anti‐rabbit IgG‐Alexa 488 (Invitrogen, Karlsruhe, Germany), and anti‐mouse IgG‐Alexa 488 (Invitrogen). Nuclei were counterstained with Hoechst 33258 (Dojindo, Kumamoto, Japan) and images were captured using an Olympus BX51 microscope.