Mouse anti rh1 4c5

The retinas of 0-24 hr old adult flies were dissected in phosphate buffer (pH 7.4). The cornea and the rest of the head including the brain tissue were removed using forceps. Subsequently, the retinas were fixed in 4% formaldehyde in phosphate buffer (pH 7.4) for 15 min, followed by a 30 min wash in phosphate buffer (pH 7.4). Prior to antibody staining, the retinas were kept in a 0.3% Triton X-100 phosphate buffer solution at 4° for 24 hr or longer. The antibody staining was done according to a standard protocol. The following antibodies were used: rat anti-Crb (F3, 1:500; Pellikka et al., 2002 (link)); mouse anti-Rh1 (4C5, 1:50; Developmental Studies Hybridoma Bank), guinea pig anti-Eys (G5, 1:500; Husain et al., 2006 (link)), mouse anti-Nervana (nrv5f7, 1:50, Developmental Studies Hybridoma Bank), rabbit anti-GM130 (ab30637, 1:300, Abcam). Secondary antibodies anti-guinea pig alexa fluor 647 (A21450), anti-rat alexa fluor 555 (A21434), and anti-mouse alexa fluor 488 (A11029) were used at 1:400 (Molecular Probes/Thermo Fisher Scientific). Acti-stain555 (PHDG1-A, 1:75, Cytoskeleton Inc) was used to visualize rhabdomeres. A Leica TCS SP8 confocal microscope with 100x oil objective (NA 1.4) was used to capture images. Image J (Fiji) and Adobe Photoshop and Adobe Illustrator were used to edit and compile figures.

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.

Dissection of eye and whole mount preparations were performed as described previously (Satoh et al., 2005 (link)). Alexa-Fluor-488-conjugated phalloidin for F-actin localization was from Molecular Probes (A-12379, Life Technologies). Rat anti-Elav (1:50 dilution), mouse anti-Rh1 4C5 (1:50 dilution) and mouse anti-Myc 9E10 antibodies (1:50 dilution) were obtained from the Developmental Studies Hybridoma Bank. Rabbit anti-Rh1 N-terminal antibody (1:1000 dilution) was made by our laboratory (Satoh et al., 2005 (link)). Mouse monoclonal anti-GFP (clone 3E6) antibody was obtained from Life Technologies. Rabbit polyclonal anti-NinaA was a gift from Nansi Jo Colley (Department of Ophthalmology and Visual Sciences and Department of Genetics, University of Wisconsin, Madison, WI). FM4-64 for membrane staining was from Invitrogen (F34653). Secondary antibodies were anti-mouse and -rabbit-IgG, labeled with Alexa Fluor 488, 568 or 647 (Molecular Probes). Samples were mounted onto glass slides with mounting medium containing 50% glycerol in PBS and 0.25% n-propyl gallate (pH 7.5), and images were taken using a Zeiss LSM 710 confocal microscope.