Mouse anti phospho tyrosine 4g10

Embryos were fixed in a 1:4 solution of 4% paraformaldehyde: heptane for 20 min and devitellinized in methanol. For visualization of MTs, embryos were prepared as previously described (Theurkauf, 1994 (link)). Briefly, embryos were fixed in a 1:1 mixture of 37% paraformaldehyde: heptane for 3 min, rinsed in PBS, and manually devitellinized using 30G PrecisionGlide needles (BD). Embryos were blocked in BBT buffer (PBS supplemented with 0.1% BSA and 0.1% Tween-20); for Asl staining, 0.5% BSA was used. Samples were incubated overnight at 4°C with primary antibody in BBT, further blocked in BBT supplemented with 2% normal goat serum, and incubated for 2 hr at room temperature with secondary antibody with DAPI. Embryos were mounted in Aqua-Poly/Mount (Polysciences, Inc.) prior to imaging.
The following primary antibodies were used: rabbit anti-Vas (1:2000, gift from P. Lasko, McGill University), rat anti-Vas (1:10, DSHB), guinea pig anti-Asl (1:4000, gift from G. Rogers, University of Arizona), mouse anti-α-Tubulin DM1α (1:500, Sigma-Aldrich T6199), rabbit anti-phospho-Histone H3 Ser10 (pH3; 1:1000, Millipore 05–570), mouse anti-phospho-Tyrosine 4G10 (pTyr; 1:1000, Millipore 05–321), Alexa 568 conjugated phalloidin (Invitrogen #12380). Secondary antibodies: Alexa Fluor 488, 568, or 647 (1:500, Molecular Probes), and DAPI (10 ng/ml, ThermoFisher).

Mouse anti-HDAC2 3F3 ChIP grade (ab51832), rabbit anti-HDAC2 ChIP grade (ab7029) and rabbit anti-HDAC1 (ab184651) were purchased from Abcam (Cambridge, United Kingdom). Rabbit anti-acetyl-Histone H3 (06-599) were purchased from Millipore (Billerica, USA). Mouse anti-c-Abl (sc-23), rabbit anti-B-tubulin (sc-9104), mouse anti-GAPDH (sc-32233) were purchased from Santa Cruz biotechnology (Dallas, United States of America). Rabbit anti-phospho-c-Abl (C4240), Filipin, The ANTI-FLAG M2 affinity gel (A2220), methyl-β-cyclodextrin and Vitamin E ((+)-α-tocopherol acetate semisynthetic from natural α-tocopherol) was purchased from Sigma-aldrich. Rabbit anti-Calbindin D-28K (AB1778) was purchased from Chemicon International (Temecula, CA, USA). The anti-NPC1 antibody was kindly donated by Dr. William Garver (University of Arizona, Tucson, AZ). Imatinib mesylate (13139), MS275 and CI994 was purchased from Cayman Chemical Company (Ann Arbor, United States of America). Torin1 (4247) was purchased from Tocris.
GNF2 was obtained from National Center for Advancing Tranlational Sciences (Center at the National Institutes of Health (NIH). U18666A (U18) drug was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The mouse anti-phospho-tyrosine (4G10) was purchased from Millipore.

Appropriately staged larvae were crudely dissected in Drosophila Ringers buffer, and fixed in 4% formaldehyde, PBS, 0.1% TritonX-100 (PTx) for 20 min. Carcasses were washed with Ptx and then blocked for one hour in PTx containing 4% normal donkey serum. Samples were incubated overnight at 4 °C with the appropriate primary antibody mix diluted in block: Chicken anti-GFP (1:2000, Aves GF-1020), Rat Anti-C15 (1:1000, gift from G Campbell), Rat anti-E-Cadherin (1:20, DSHB), mouse anti Fasc II (DSHB), Rabbit Anti-Bazooka (1:1000, gift of A. Wodarz, GST N-term (Wodarz et al., 1999 (link))), Mouse anti-phospho tyrosine 4G10 (1:1000, Millipore); rabbit anti-Zipper (1:1000, gift of Dan Kiehart); mouse anti-FLAG (1:5000, Sigma F3165). Secondary antibodies were labeled with Alexa488, Cy3, Cy5 or Alexa 690.
Images were acquired on a Zeiss Imager with Apotome using a 40×, 1.2 NA lens, or using a Leica confocal system with Yokogawa CSU-X1 spinning disk, using either a water immersion 40×, (NA. 1.1) or 63 (1.2), or an oil imersion 100× (1.3) lens, driven by Metamorph (v7.8.4).