Cy5 conjugated secondary antibody

The plasmatocytes that were recruited to the brains or in circulation were immunostained with anti-NimC1 and anti-pFAK antibodies. Circulating hemocytes were collected from the transgenic flies by cutting heads out of the bodies to exude the hemolymph. The collected hemocytes were plated on 0.01% poly-L-lysine-coated coverslips (12 × 12 mm) for 40 min, fixed (4% paraformaldehyde) for 20 min and permeabilized (PBS buffer with 0.2% Triton x-100) for 10 min, followed by blocking in bovine serum (PBS buffer with 0.2% Triton x-100 and 2% BSA) for 30 min at room temperature (RT). The hemocytes were then immunostained with anti-NimC1 or anti-pFAK antibodies at 4 °C overnight. After washing, the cells were incubated with FITC or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, 1:200) at 4 °C overnight and then counterstained with DAPI (1:500) at RT for 30 min prior to mounting for confocal microscopy. The dissected brains were fixed with 4% paraformaldehyde for 50 min at RT. After washing with PBS buffer, brains were permeabilized (PBS buffer containing 0.1% sodium citrate and 0.1 % Triton-x-100) for 30 min at RT and subsequently incubated with blocking buffer (washing buffer containing 2% BSA) for 1 h at 4 °C prior to performing immunostaining for anti-NimC1 and anti-pFAK. Quantification of the fluorescence intensities of NimC1 were determined using ImageJ.

Confocal microscopy analyses were carried out as previously described [59 (link)]. In brief, twenty-four hours after infection, cells were fixed with 4% paraformaldehyde (CARLO ERBA Reagents, 387507) in PBS followed by permeabilization with 0.2% triton X-100 (Sigma-Aldrich, T9284) in PBS. Cells were labeled with the primary antibody anti-LC3 (Cosmo Bio Ltd, CTB-LC3-2-IC), anti-NDP52 (Ab-184688, Abcam), anti-Ubiquitin (FK2) (ST1200, Millipore) for 1 h at room temperature and visualized by means of Cy5-conjugated secondary antibodies (Jackson Immunoresearch) or Alexa Fluor-488 secondary antibodies (Thermo Fisher Scientific). Coverslips were mounted in Prolong Gold antifade (P36935, Life Technologies) and examined under a confocal microscope (Leica TCS SP2). Digital images were acquired with the Leica software and studies of colocalization were performed by using appropriated tools of ImageJ software [60 (link)]. A minimum of 200 cells per sample experimental condition were counted for triplicate samples per condition in each experiment.

Immunostaining of the pupal eye was carried out as described [14] (link). Rat anti-Kirre (1∶5000) and rabbit anti-Hbs AS14 (1∶2500) were used as previously described [23] (link). Other primary antibodies: mouse anti-Rst Mab24A5.1(1∶100) [33] (link), rabbit anti-Sns (1∶300) [34] (link) and rabbit anti-lacZ (1∶2000; 5 Prime→3 Prime). Rat anti-DE-cadherin (1∶20), mouse anti-Armadillo (1∶20) and mouse anti-Dl 9B (1∶20) were provided by Developmental Studies Hybridoma Bank at the University of Iowa. Secondary antibodies: Alexa 488 and Alexa 568 conjugated secondary antibodies (1∶5000; Molecular Probes); Cy5 conjugated secondary antibodies (1∶1000; Jackson ImmunoResearch Laboratories). All images were captured using an Axioplan2 epi-fluorescence microscope equipped with an Axiocam digital camera (Carl Zeiss, Inc.).