Immunostaining of Drosophila Visual System

Immunostaining of imaginal discs were performed with standard protocols.⁵⁶ (link)^,⁵⁷ (link) To immunostain the pupal Drosophila visual system, samples should be dissected after 48h when third-instar larvae begin to enter pupation. Then the whole visual system is held in fixative and primary antibody solution with shaking in overnight at 4°C. After adding the secondary antibody, the Drosophila pupal visual system can be mounted for confocal microscopy (TCS SP5II, Leica Company, Germany). Antibodies were used in this study: rat anti-Ci (2A) (1:50; DSHB); mouse anti-Flag (M2) (1:200; Sigma); mouse anti-HA (F7) (1:200; Santa Cruz); TRITC-labeled phalloidin (1:250; Sigma).

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Gao Y., Shan Z., Jian C., Wang Y., Yao X., Li S., Ti X., Zhao G., Liu C, & Zhang Q. (2023). HIB/SPOP inhibits Ci/Gli-mediated tumorigenesis by modulating the RNA Polymerase II components stabilities. iScience, 26(8), 107334.

Publication 2023

mouse anti-Flag (M2) mouse anti-HA (F7) TRITC-labeled phalloidin

Antibodies Antibody Confocal microscopy Drosophila Fixative Held Imaginal discs Larvae Mouse Pupal Tritc phalloidin

Corresponding Organization : Nanjing Medical University

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Variable analysis

independent variables

Time of dissection (48h after third-instar larvae begin to enter pupation)

dependent variables

Localization and expression of proteins (Ci, Flag, HA) in the Drosophila pupal visual system

control variables

Standard protocols used for immunostaining of imaginal discs
Fixation and primary antibody incubation conditions (shaking in overnight at 4°C)
Secondary antibody incubation
Mounting for confocal microscopy

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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