Image axio vision

Fat bodies were dissected from the control and bacteria infected individuals as described in section 4.3. The tissue was further fixed for 15 minutes in 4% paraformaldehyde at room temperature and mounted on a microscope slide using RotiMount Flour-Care DAPI (#HP20.1, Carl Roth GmbH, Karlsruhe, Baden-Württemberg, Germany). The hemocytes were collected using a microcapillary as described earlier and the hemolymph volume was released onto a microscope slide coated with Poly-L-Lysine. This was immediately followed by putting a small drop of PBS over the hemolymph and incubating the slide in a moist chamber for 15 minutes, to let the cells adhere to the slide. The cells were further fixed using 4% Paraformaldehyde for 10 minutes, gently washed with 0.1% PBST (PBS containing 0.1% Tween 20) three times and mounted using Rotimount. Imaging was performed using fluorescence microscope equipped with Apotome or by using a CLSM880 (Carl Zeiss Image Axio Vision, Jena, Germany).

Immunohistochemistry was performed as previously described (Li et al., 2016 (link)). The brains were dissected in Drosophila Ringer’s solution and immediately fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Subsequently, the samples were washed with PBST (0.3% Triton X-100 in PBS) and blocked in blocking-buffer (10% goat serum in PBST) for 30 min at room temperature, followed by incubation with the primary antibody (1:200 rabbit anti-dILP2, a gift from Eric Rulifson, UCSF, USA) overnight at 4°C with subsequent application of the secondary antibody (1:500 donkey anti-rabbit IgG, Jackson ImmunoLabs, Suffolk, UK) for 3 h at room temperature. After three washings, the brains were mounted on slides and images were obtained using a fluorescent microscope equipped with an apotome (Carl Zeiss Image AxioVision, Göttingen, Germany). To facilitate the quantification of dILP2 fluorescence intensities in the region of pars intercerebralis, series of sections were gathered under identical thickness, exposure time and all other relevant settings. Fluorescence intensity was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).