Immunostaining of Drosophila Larval Brains

Larvae of desired genotype were dissected at 96 h ALH and brains were fixed for 15 min in 3.7% formaldehyde in PBS with 0.1% Triton-X, and subsequently processed for immunochemistry analysis. The following antibodies were used: mouse anti-Flag (Sigma, F1804), 1/2000; rabbit anti-Mira^{68 (link)} (generated in our lab), 1/1000; guinea-pig anti-Dpn (ref. ^{68 (link)}; generated in our lab), 1/1000; rabbit anti-Baz (generated in our lab), 1/2000; rabbit anti-Par6 (generated in our lab), 1/1000; rabbit anti-aPKCζ C20 (Santa Cruz Biotechnologies, SG-216-G), 1/1000; chicken anti-GFP (Abcam, ab13970), 1/5000; rabbit anti-Pon (generated in our lab), 1/2000; and rabbit anti-Numb (a gift from Xiaohang Yang), 1/1000. Secondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 633 (Molecular Probes) used at 1/500, 1/1000, and 1/250, respectively. TO-PRO-3 (Invitrogen) or Hoechst 33342 (Sigma) was used at 1/5000 for DNA staining, and samples were mounted in Vectashield (Vector Laboratories). Images were obtained using Leica SP8 confocal microscope or Zeiss LSM 780 laser scanning confocal microscope and processed in Adobe Photoshop CC2018.

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Liu Z., Yang Y., Gu A., Xu J., Mao Y., Lu H., Hu W., Lei Q.Y., Li Z., Zhang M., Cai Y, & Wen W. (2020). Par complex cluster formation mediated by phase separation. Nature Communications, 11, 2266.

Publication 2020

mouse anti-Flag rabbit anti-aPKCζ C20 chicken anti-GFP Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 633 Hoechst 33342 Vectashield SP8 confocal microscope

Alexa fluor 488 Alexa fluor 555 Antibodies Brains Chicken Confocal microscope Formaldehyde Genotype Guinea pig Hoechst 33342 Larvae Laser scanning microscope Molecular probes Mouse Rabbit Vector

Corresponding Organization :

Other organizations : Huashan Hospital, Fudan University, Temasek Life Sciences Laboratory, National University of Singapore, Fudan University Shanghai Cancer Center, Capital Normal University, Hong Kong University of Science and Technology, University of Hong Kong

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