Roti mount flour care dapi

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).

Huh7 cells were cultured in 4-well chamber slides (Nunc Lab-Tek, SigmaAldrich). 24 h after transfection with pcDNA4/To/myc-His B-IFNλ4 plasmid or with pcDNA4/To/myc-His B vector, using PEI, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 in PBS containing 2% BSA. Fixed and permeabilized cells were incubated with anti-IFNλ4 (Merck Millipore, 5 μg/ml) and anti-Giantin antibodies (Abcam,1 μg/ml) in PBS containing 2% BSA for 1 h at 37 °C. After three times washing with PBS, the corresponding secondary antibodies (goat anti-rabbit 647, 1:1000, and goat anti-mouse 488, 1:400) were added under the same conditions as the primary antibodies. Excess antibodies were removed by three times washing with PBS. Endoplasmic reticulum (ER) staining was performed with the Cytopainter ER staining Kit-Red Fluorescence (Abcam) for 30 m at 37 °C. Slides were mounted with Roti-mount FlourCare DAPI (Carl Roth). Confocal microscopy was performed with a Zeiss LSM 710 confocal microscope and images were acquired with the ZEN 2010 software (Carl Zeiss). The colocalization coefficient (Pearson’s coefficient) was quantified in ImageJ using the plug-in, JACoP (Just Another Colocalization Plug-in)^{47 (link)}.

Air-dried cryostat sections (4 μm thickness) were fixed (methanol, 8 min). Unspecific binding sites were blocked in 2% BSA (Roth, Karlsruhe, Germany) for 1 h followed by incubation with 1 μg of the following AlexaFluor488, AlexaFluor594, and AlexaFluor 647-labeled mAbs: CD4, CD8α, CD11b, Gr1, CD11c, F4/80, CD104, CD206, PD-1, and PD-L1 (all from Biolegend). For intracellular stainings, slides were fixed in 4% paraformaldehyde w/o methanol (Thermo Scientific, Darmstadt, Germany, 30min) and cells permeabilized in 0.5% Triton X−100 (Sigma-Aldrich, Darmstadt, Germany, 15 min). After blocking with 2% BSA (Serva, Heidelberg, Germany), slides were incubated with the monoclonal rabbit anti-IRF5 antibody (1:50; ThermoFisher Scientific, Darmstadt, Germany) overnight at 4 °C, followed by a secondary goat anti-rabbit Alexa647 antibody (1:500; Cell Signaling, Frankfurt am Main, Germany). Sections were washed, embedded in Roti Mount Flour Care DAPI (Roth), and target proteins visualized on a confocal laser scanning microscope (Elyra 7, Zeiss, Jena, Germany) using 20× objectives. IRF5⁺ cells were quantified by counting individual positive cells in three high power fields per sample (n = 3/group).