Immunohistochemical Staining Protocol

Tissue dissections were performed in PBS followed by fixation in 4% PFA for 20 minutes at room temperature. For this study, the following primary antibodies were: mouse anti-FLAG M5 (1:1,000) (Sigma); rabbit anti-Phm (1:200) [30 (link)], guinea pig anti-Spok (1:200) [61 (link)]; guinea pig anti-Sro (1:1,000) [62 (link)]. Tissues were incubated over night with primary antibodies at 4°C. Fluorescent conjugated secondary antibodies used in this study, goat anti-mouse Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555 and goat anti-guinea pig Alexa Fluor 555, were purchased from Life Technologies. Secondary antibodies were diluted 1:500 and incubated for 1 hour at room temperature. Confocal images were captured using Carl Zeiss LSM 700 laser scanning microscope.

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Komura-Kawa T., Hirota K., Shimada-Niwa Y., Yamauchi R., Shimell M., Shinoda T., Fukamizu A., O’Connor M.B, & Niwa R. (2015). The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier. PLoS Genetics, 11(12), e1005712.

Publication 2015

mouse anti-FLAG M5 goat anti-mouse Alexa Fluor 488 goat anti-guinea pig Alexa Fluor 488 goat anti-rabbit Alexa Fluor 555 LSM 700 laser scanning microscope

Alexa fluor 488 Alexa fluor 555 Antibodies Fluorescent antibodies Goat Guinea pig Laser scanning microscope Mouse Rabbit Tissue dissections Tissues

Corresponding Organization : Japan Science and Technology Agency

Other organizations : University of Minnesota, Institute of Agrobiological Sciences

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Variable analysis

independent variables

Primary antibodies used: mouse anti-FLAG M5, rabbit anti-Phm, guinea pig anti-Spok, guinea pig anti-Sro

dependent variables

Fluorescent signals from antibody labeling

control variables

Tissue dissection and fixation protocol (PBS, 4% PFA, 20 minutes at room temperature)
Incubation conditions for primary and secondary antibodies (overnight at 4°C, 1 hour at room temperature)
Imaging method (confocal microscopy)

controls

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

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