Anti mouse cy3

Ovaries from 3 to 4 day old females were dissected in PBS and fixed for 20 min at room temperature (RT) in 4% paraformaldehyde in PBS, pre-absorbed in 2% BSA, 0.3% Tween-20 or Triton X-100 in PBS for 1 h at RT, incubated with primary antibodies overnight 4 °C, secondary antibodies for 2 h at RT, stained with 1 µg/ml Hoechst 33342 (Molecular Probes) for 10 min at RT, and ovarioles were separated and mounted in 80% glycerol 0.5% N-propylgalate mounting media. Antibody dilutions: mouse monoclonal anti-Lamin C (DSHB, LC28.26) 1:200, mouse monoclonal anti-Fasciclin 3 (DSHB, 7G10) 1:40, mouse monoclonal Eyes Absent (DSHB, eya10H6) 1:200, rabbit polyclonal anti-GFP (Life Technologies), anti-mouse-Cy3 (Life Technologies) 1:200, and anti-rabbit-Alexa Fluor 488 1:200 (Cell Signaling). To detect apoptosis, ovaries stained with Lamin C were fixed a second time (4% paraformaldehyde, PBS) prior to ApopTag^® Red In situ Apoptosis Detection Kit (Milipore). Ovaries were incubated in equilibration buffer 10 s RT, 1 h 37 °C in Tdt enzyme solution, 10 min RT in stop/wash buffer, washed 0.3% Tween-20, PBS, and Rhodamine antibody solution for 30 min at RT. Confocal images were taken using a Zeiss 510Meta LSM or Zeiss 800 with multiple consecutive Z-sections (0.2–0.3 mm per slice), processed with ImageJ (1.46 R) and annotated using Adobe Photoshop CS5.1 and Adobe Illustrator CC 2015.

For subcellular localization, we co-expressed the MoPrP and CD8-GFP as a cell membrane marker in interneurons of the larval ventral ganglion under the control of OK107-Gal4. Whole-mount immunohistochemistry of fixed larval brains was conducted by fixing in 4% formaldehyde, washing with PBT, and blocking with 3% bovine serum albumin before incubating with the primary antibody as described previously (Fernandez-Funez et al., 2010 (link)). We incubated first with the 6H4 anti-PrP antibody (1: 1,000) followed by the secondary antibody anti-mouse-Cy3 (Molecular Probes) at 1:600. We mounted the stained larval brain in Vectashield antifade (Vector) mounting medium for microscopic observation and documentation. We collected two-channel fluorescent images with AxioVision (Zeiss) in an Axio- Observer Z1 microscope (Zeiss) by optical sectioning using ApoTome (structured light microscopy) with a 63x NA: 1.4 (oil) objective. Representative single plane images of local interneurons were extracted from Z-stacks. Final images were combined using Adobe Photoshop and processing included brightness/contrast adjustment to whole images for optimal viewing and printing.

For subcellular localization, we co-expressed the PrP constructs and CD8-GFP as a membrane marker in intemeurons of the larval ventral ganglion under the control of OK107-Gal4 (UAS-CD8-GFP; OK107-Gal4/UAS-PrP). Whole-mount immunohistochemistry of fixed larval brains was conducted by fixing in 4% formaldehyde, washing with PBT, and blocking with 3% bovine serum albumin before incubating with the primary antibody as described previously (Fernandez-Funez et al., 2010). We incubated first with the 6H4 anti-PrP antibody (1: 1,000) followed by the secondary antibody anti-mouse-Cy3 (Molecular Probes) at 1:600. We mounted the stained larval brain in Vectashield antifade (Vector) mounting medium for microscopic observation and documentation. Images of single neurons were taken at and 630x (see details below).
We collected fluorescent images by optical sectioning using AxioVision (Zeiss) software in an Axio-Observer Z1 microscope (Zeiss) equipped with ApoTome (structured light microscopy) with 40x NA: 1.3 (air) and 63x NA: 1.4 oil objectives. Images were combined using Adobe Photoshop; processing included trimming of non-informative edges and brightness / contrast adjustment to whole images.