Immunochemistry Analysis of 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 later processed for immunochemistry analysis. The following antibodies were used: mouse anti-Mira (Fumio Matsuzaki), 1/30; mouse anti-flag (Sigma), 1/2,000; rabbit anti-Mira49 (link) (generated in our lab), 1/1,000; rabbit anti-Stau (Daniel St Johnston), 1/2,000; rat anti-Brat50 (link) (Yongqing Zhang), 1/500; mouse anti-Pros (DSHB), 1/20; guinea-pig anti-Dpn (generated in our lab), 1/1,000; rabbit anti-Pon (generated in our lab), 1/2,000; rabbit anti-aPKCζ C20 (Santa Cruz Biotechnologies), 1/1,000; chicken anti-GFP (Abcam), 1/5,000. Secondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 633 (Molecular Probes), and used at 1/500, 1/1,000, and 1/250, respectively. TO-PRO-3 (Invitrogen) was used at 1/5,000 for DNA staining and samples were mounted in Vectashield (Vector Laboratories). Images were obtained using Zeiss LSM 510 upright or Leica SP II upright microscope and processed in Adobe Photoshop CS6 and Adobe Illustrator CS6.

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Jia M., Shan Z., Yang Y., Liu C., Li J., Luo Z.G., Zhang M., Cai Y., Wen W, & Wang W. (2015). The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division. Nature Communications, 6, 8381.

Publication 2015

mouse anti-flag rabbit anti-aPKCζ C20 chicken anti-GFP Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 633 Vectashield Illustrator CS6

Alexa fluor 488 Alexa fluor 555 Antibodies Brains Chicken Formaldehyde Genotype Guinea pig Larvae Microscope Molecular probes Mouse Rabbit Vector

Corresponding Organization :

Other organizations : Fudan University, Temasek Life Sciences Laboratory, National University of Singapore, Hong Kong University of Science and Technology, University of Hong Kong, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

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Variable analysis

independent variables

Genotype of the larvae

dependent variables

Localization and expression patterns of the following proteins: Mira, Flag, Mira49, Stau, Brat50, Pros, Dpn, Pon, aPKCζ

control variables

Dissection time at 96 h ALH
Fixation time of 15 min in 3.7% formaldehyde in PBS with 0.1% Triton-X
Antibody concentrations: mouse anti-Mira (1/30), mouse anti-flag (1/2,000), rabbit anti-Mira49 (1/1,000), rabbit anti-Stau (1/2,000), rat anti-Brat50 (1/500), mouse anti-Pros (1/20), guinea-pig anti-Dpn (1/1,000), rabbit anti-Pon (1/2,000), rabbit anti-aPKCζ C20 (1/1,000), chicken anti-GFP (1/5,000)
Secondary antibody concentrations: Alexa Fluor 488 (1/500), Alexa Fluor 555 (1/1,000), Alexa Fluor 633 (1/250)
TO-PRO-3 concentration (1/5,000) for DNA staining
Mounting medium: Vectashield
Imaging equipment: Zeiss LSM 510 upright or Leica SP II upright microscope

positive controls

None specified

negative controls

None specified

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