Goat anti rabbit cy5

Intracellular cytokine staining was performed as described before [62] (link) with modifications. Differentiated macrophages (∼1×10⁷cells/plate) were washed and challenged with L. pneumophila GFP⁺ strains in RPMI containing 200 µg/mL thymidine, 5 µg/mL Chloramphenicol and 1 mM IPTG. For measuring TNF production, infections were carried out at MOI-3 and MOI-10 for 9 hrs followed by Golgiplug incubation (BD Bioscience, 1 µL/mL) for additional 5 hrs. For IL-1α and IL-1β, infections were carried out for 6 hrs. Cells were harvested with 10 mL cold PBS, washed twice with FACS buffer (PBS+0.5%BSA+0.05%NaN), incubated with Fc Block (clone 2.4G2) for 20 minutes and fixed with 2% paraformaldehyde overnight at 4°. Macrophage were permeabilized with Perm/Wash buffer (BD Bioscience) on ice for 20 minutes and stained with Alexa Fluor 647-conjugated anti-mouse TNF (BioLegend, clone ALF 161), phycoerythrin(PE)-conjugated anti-mouse IL-1α (BioLegend, clone MP6-XT22), PE-conjugated anti-mouse IL-1β (eBioscience, clone NJTEN3), Rabbit anti-DUSP1 (MKP1 V-15, Santa Cruz) and Goat anti-Rabbit Cy5 (Invitrogen) for 40 minutes. Stained cells were analyzed by BD LSR II flow cytometer.

Adult abdominal and larval muscles were dissected in saline and fixed with 4% paraformaldehyde for 15 min or Bouin's solution for 10 min. The dissected muscles were washed with 2% PBS-Triton X-100 and incubated with rabbit polyclonal antibody Ref(2)p/p62 (rabbit pAb, Abcam, ab178440) at a 1:500 dilution, TDP-43 rabbit polyclonal antibody (pAb, Proteintech, 10782-2-Ap) at a 1:500 dilution or Lamin C mouse monoclonal antibody (mAb, Developmental Studies Hybridoma Bank, LC28.26) at a 1:100 dilution overnight at 4°C on a nutator. Subsequently, the muscles were washed three times with 2% PBS-Triton X-100 and incubated overnight at 4°C with either goat anti-rabbit-Cy5 or goat anti-mouse-Alexa 555 secondary antibodies (Invitrogen) at a 1:500 dilution. Samples were mounted on a glass slide with Vectashield antifade mounting medium and imaged using a Leica DMi8 microscope with a 100× objective (1.4NA) and THUNDER imager.

The anti-AVT antibody was a generous gift from Dr Soojin Ryu (Johannes Gutenberg University, Mainz, Germany). Immunofluorescence labelling was carried out according to standard procedures. Brains were dissected fresh and fixed in 4% PFA for 2 days at 4 °C, and were then washed in phosphate buffered saline (PBS) and stored in methanol at −20 °C until processing. 100 μm coronal sections were collected using a vibratome (Leica VT1000 S, Leica Biosystems). After blocking in PBS containing 5% normal goat serum (Sigma, Cat. no. G9023), 1% dimethyl sulfoxide (Sigma, Cat. no. 276855) and 0.2% Triton X-100 (Fisher, Cat. no. 10254640), we incubated in primary antibody (Rabbit anti-AVT, 1:500^{38 (link)}) for 24 h at 4 °C. The secondary antibody (Goat anti-rabbit Cy5, 1:500; Invitrogen, Cat no. A10523) was incubated for 2 h at room temperature. Brain sections were imaged at the level of the preoptic area using an Olympus FV1000 confocal microscope with a 20x Nikon objective. Images were assembled using Amira software (Thermo Scientific).