Phalloidin cy5

Immunostaining was performed as described previously (Shlevkov and Morata, 2012 (link)). Images were captured with Leica TCS SPE and Zeiss LSM510 Vertical confocal microscopes. Images were processed with Fiji-ImageJ and Adobe Photoshop CS4 software using Student’s t for significance tests. Statistical analyses were performed with Microsoft Excel software. The following primary antibodies were used: rabbit anti-Caspase 3 (Roche, Basilea, Switzerland) 1:50; mouse anti-ß-Galactosidase (Promega, Madison, WI); rat anti-Cubitus interruptus (2A1; Hybridoma Bank, Iowa City, IA) 1:25; mouse anti-Engrailed (4D9; Hybridoma Bank) 1:50; mouse anti-Patched (Apa-1; Hybridoma Bank) 1:50; rabbit anti-H3K4me3 (07-473; Millipore, Billerica, MA) 1:50. Fluorescently labeled secondary antibodies (Molecular Probes, Eugene, OR) were used in a 1:200 dilution. TO-PRO3 (Invitrogen, Carlsbad, CA) was used in a 1:600 dilution to label nuclei; Phalloidin-Cy5 (65906; Sigma, St. Louis, MO) was used in a 1:200 dilution to label the F-actin network (thus the cell membranes).

To quantify the number of mCherry E. coli bacteria (K12 E. coli expressing PDSpRSETD-cherry plasmid from Dr. Stephan Mesnage) in hemocytes in vivo, He-Gal4; UAS-FYVE-GFP or he-Gal4; UAS-FYVE-GFP, hemo^A4 flies were injected with 0.2 μL of bacterial culture grown to OD600 = 0.05, using a glass capillary microinjection needle. Flies were then dissected as described in Magny et. al 2013 [7 (link)] to expose the dorsal abdominal cuticle in order to image the hemocytes associated to the dorsal vessel, as described in Horn et al. 2014 [71 (link)]. Flies were dissected after 20 or 120 min postinjection, in PBS, and the cuticles fixed in 4% PFA in PBS for 20 min. The preparations were then washed in, PBTX, incubated 30 min in PBS with phalloidin-Cy5 (Sigma-Aldrich) (1:10), washed in PBS, and mounted in Vectashield (vector). Hemocytes were imaged with a Zeiss laser scanning microscope LSM 5.10 on a Zeiss Axioskop 2 stage, with a 40X Achroplan objective, and bacterial cells within hemocytes, as determined with the phalloidin and FYVE counter-stains, were counted in Z-stack reconstructions.