Immunohistochemical Analysis of Drosophila Antennae and Testes

Embryo and pupal antenna immunohistochemistry was carried out according to (zur Lage et al., 2018 (link)). The fixing and staining of Drosophila testis was described in Sitaram et al., 2014 (Sitaram et al., 2014 ). The following primary antibodies were used: rabbit anti-GFP antibody (1:500, Life Technologies, A11122), mouse anti-Futsch antibody (1:200, Developmental Studies Hybridoma Bank, 22C10-s), mouse anti-acetylated tubulin (1:1000, Sigma, T6793) and rabbit anti-Dnah5 antibody (1:2000, see below) and the secondary antibodies were goat anti-Rabbit antibody (1:500, Alexa Fluor 488, Life Technologies, A11008) and goat anti-Mouse antibody (1:500, Alexa Fluor 568, Life Technologies, A11019). DNA in adult testes was stained with To-Pro-3 (1:1000, Life Technologies, T3605) solution in the dark for 15 min. After several washes, the samples were mounted on slides with 85% glycerol and 2.5% propyl gallate (Sigma-Aldrich, P3130). The slides were imaged using a Zeiss LSM-5 PASCAL/Axioskop 2 confocal microscope and processed with Fiji.

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zur Lage P., Xi Z., Lennon J., Hunter I., Chan W.K., Bolado Carrancio A., von Kriegsheim A, & Jarman A.P. (2021). The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly. Biology Open, 10(10), bio058812.

Publication 2021

rabbit anti-GFP antibody Alexa Fluor 488 Alexa Fluor 568 P3130

Adult Alexa 568 Alexa fluor 488 Anti antibody Antibodies Confocal microscope Drosophila Embryo Glycerol Goat Hybridoma Immunohistochemistry Mouse Propyl gallate Pupal Rabbit Testes Tubulin

Corresponding Organization :

Other organizations : University of Edinburgh, Edinburgh Cancer Research

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Fixing and staining methods used for Drosophila testis

dependent variables

Embryo and pupal antenna immunohistochemistry results
Drosophila testis characteristics

control variables

Primary antibodies used: rabbit anti-GFP, mouse anti-Futsch, mouse anti-acetylated tubulin, and rabbit anti-Dnah5
Secondary antibodies used: goat anti-Rabbit (Alexa Fluor 488) and goat anti-Mouse (Alexa Fluor 568)
DNA staining in adult testes with To-Pro-3
Mounting medium used: 85% glycerol and 2.5% propyl gallate

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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