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Immunological analysis of glycosylation-related proteins in Drosophila tissues
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Immunological analysis of glycosylation-related proteins in Drosophila tissues

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Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

Immunological analysis of glycosylation-related proteins in Drosophila tissues

Authors
 Yamamoto-Hino, Miki
Department of Physiology, School of Medicine, Keio University

Okano, Hideyuki
Department of Physiology, School of Medicine, Keio University

Goto, Satoshi *
Department of Life Science, Rikkyo University
*To whom correspondence should be addressed.
KeyWords
Reagents
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PBS: phosphate buffered saline

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Fixative: 4% paraformaldehyde in PBS

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Washing solution (PBST): 0.1% TritonX-100 or Tween20 in PBS

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Blocking solution: 0.5% BSA (Bovine serum albumin) in PBST

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Antibodies: anti-GM130, anti-dGLG1, anti-Syx16, anti-rabbit IgG, anti-rat IgG

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Mounting medium: vectashield (Vector Laboratories, Inc., Burlingame, CA)

Methods
1.

Fixation of Drosophila tissues

1) 

 Dissect Drosophila larvae or adults in PBS.

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2) 

 Fix tissues in fixative at room temperature for 20–40 min.

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3) 

 Wash tissues in washing solution for 10 min three times.

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2.

Immunostaining of Drosophila tissues

1) 

 Block tissues with blocking solution at room temperature for 30 min.

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2) 

 Incubate tissue with the appropriate first antibody at 4°C for more than 8 h.

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3) 

 Wash tissues in washing solution for 10 min three times.

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4) 

 Incubate tissue with the appropriate secondary antibody at 4°C or room temperature for 2 h or for more than 4 h, respectively.

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3.

Observation of Drosophila tissues

1) 

 Mount stained tissue with mounting medium.

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2) 

 Observe of tissue through a laser scanning confocal microscopy.

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Discussion

In Drosophila cells, anti-GM130, anti-dGLG1 and anti-Syx16 clearly stained cis-, medial- and trans-cisternae, respectively (Yano et al., 2005; Yamamoto-Hino et al., 2012). Co-staining using antibodies against these cisternal markers and proteins or tagged proteins of interest reveals in which cisternae the proteins of interest are localized.

Figure & Legends

Figure & Legends

 

Fig. 1. Stacks of Golgi cisternae in Drosophila disc cells.

GM130 (blue), dGLG1 (red) and PNA-reactive glycans (green) are localized to cis-, medial- and trans-cisternae of the Golgi complex in Drosophila disc cells.

This figure was originally published in Proc. Natl. Acad. Sci. Yano H,  Goto S et al. "Distinct functional units of the Golgi complex in Drosophila cells" 2005, 102(38):13467-72 ©the National Academy of Sciences.

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Date of registration：2014-05-27 13:16:47
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