Drosophila Whole Mount and Tissue Section Imaging

For whole mount staining, we dissected fly tissue samples in ice cold PBS and fixed them with 4% paraformaldehyde at room temperature for 30 min as described [6] (link). For tissue sections, we fixed fly tissues with 4% paraformaldehyde, dehydrated the tissues with acetone, and embedded them in LR white resin (Electron Microscopy Sciences). Images were captured with a Zeiss LSM 710 confocal microscope. Antibodies were used at the following concentrations: rabbit anti-Rab5 (abcam), 1∶500; rabbit anti-Avl [84] (link), 1∶500; rabbit anti-Rab7 [76] (link), 1∶500; mouse anti-PDI (abcam), 1∶100; rabbit anti-GM130 (abcam), 1∶500; rabbit anti-Rab11 (abcam, [6] (link)), 1∶500; chicken anti-GFP (abcam), 1∶1,000; mouse anti-Rh1 [4C5, Developmental Studies Hybridoma Bank (DSHB)], 1∶50; mouse anti-Wash (P3H3, [131] (link)), 1∶20; guinea pig anti-Vps26, 1∶1,000 (this study); biotin-conjugated PNA (Vector Labs), 1∶1,000; Alexa 488-conjugated phalloidin (Invitrogen), 1∶100; and Alexa 405-, Alexa 488-, Cy3-, or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch), 1∶600.

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Wang S., Tan K.L., Agosto M.A., Xiong B., Yamamoto S., Sandoval H., Jaiswal M., Bayat V., Zhang K., Charng W.L., David G., Duraine L., Venkatachalam K., Wensel T.G, & Bellen H.J. (2014). The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration. PLoS Biology, 12(4), e1001847.

Publication 2014

LR white resin LSM 710 confocal microscope rabbit anti-Rab5 mouse anti-PDI rabbit anti-GM130 rabbit anti-Rab11 chicken anti-GFP mouse anti-Rh1 [4C5 biotin-conjugated PNA Alexa 488-conjugated phalloidin

Acetone Antibodies Biotin Chicken Cold Confocal microscope Electron microscopy Guinea pig Hybridoma Lr white resin Mouse Paraformaldehyde Phalloidin Rabbit Tissues Vector

Corresponding Organization :

Other organizations : Baylor College of Medicine, The University of Texas Health Science Center at San Antonio

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Variable analysis

independent variables

Dissection method (whole mount vs. tissue sections)
Fixation method (paraformaldehyde vs. acetone dehydration and LR white resin embedding)

dependent variables

Staining patterns and localization of the following proteins: Rab5, Avl, Rab7, PDI, GM130, Rab11, GFP, Rh1, Wash, Vps26

control variables

Incubation time for paraformaldehyde fixation (30 minutes)
Antibody concentrations

controls

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

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