Vectashield

Frozen sections prepared identical to those used for spatial transcriptomics (see below) were used to visualize Muc5ac. Slides stored at −80°C were warmed to room temperature and washed twice in PBS (0.37 M NaCl, 27 mM KCl, 100 mM Na₂HPO₄, and 18 mM KH₂PO₄), then fixed in −20°C methanol for 30 min. Two more washes in PBS were performed, and then the tissue was blocked using 10% Horse Serum for 30 min. Next, two washes in PBS were followed by immunolabeling with Muc5ac (Cat. no. 1364248; U.S. Biotech) and ECAD (Cat. no. AF748; R&D Systems) overnight at 4°C. Two more washes in PBS were performed the following day, and fluorescently conjugated secondary antibodies were used to detect primary antibodies (Donkey anti-Rabbit 555 703-165-155; Jackson Labs, Donkey anti-Goat A11055; Life Tech) for 1 h and 30 min at room temperature. After two washes in PBS, DAPI (Cat. no. 62248; Thermo Fisher Scientific) was applied in the final wash step at 0.1 μg/ml to label nuclei for 15 min in PBS, and slides were coverslipped and mounted in Vectashield (Cat. no. H-1000; Vector Labs). Images were acquired using Leica SP8 confocal microscope and images were processed with Leica software. Tissue was imaged in most distal airways where mucin secretion is uncommon.

Immunohistochemistry was performed on 3- to 7-day-old flies. Flies were fixed with 4% (v/v) paraformaldehyde with 0.5% Triton X-100 for 2 hours and 40 min at room temperature. Brains were dissected in phosphate-buffered saline with 0.5%Tween 20 (PBST) and then washed twice (10 min) in 0.5% PBST buffer with rotation. The 10% normal goat serum (Jackson ImmunoResearch) was used for blocking overnight at 4°C. Mouse anti-TH at 1:1000 dilution, rat anti-TIM at 1:200 dilution, and chicken anti-GFP antibody at a 1:1000 were used as primary antibody and incubated with the brains overnight at 4°C, and the brains were then washed twice (10 min) in 0.5% PBST buffer at room temperature. The corresponding secondary antibodies were added and incubated overnight at 4°C. Brains were mounted in VECTASHIELD (Thermo Fisher Scientific) and imaged on a Leica SP5 confocal microscope. The images were processed by ImageJ.

Fat bodies were dissected in ice-cold PBS and fixed in 4% paraformaldehyde at room temperature with gentle shaking for 15 min and washed in PBS for 15 min. Subsequently, the tissues were incubated with BODIPY493/503 (1:1000, D3922, Invitrogen, Eugene, USA) or Nile Red (1:1000, 72485, Sigma, St. Louis, USA) for 30 min at room temperature with gentle shaking in the dark and then washed in PBS for 30 min. Samples were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, USA). For rabbit anti-GFP (A11122, 1:2000, Invitrogen) and HA-Tag (C29F4) rabbit mAb (1:8000, #3724, Cell Signaling, Danvers, USA) staining, tissues were incubated with the primary antibody overnight at 4 °C, washed in PBS for 30 min, followed by the secondary antibody goat anti-rabbit Alexa 647 (A21245, 1:200, ThermoFisher, Eugene, USA) or goat anti-rabbit Alexa 488 (A21206, 1:200, ThermoFisher) for 1 h, washed in PBS for 30 min and finally mounted in Vectashield containing DAPI. Confocal images were acquired using Zeiss LSM 800 confocal microscope with a 63X oil objective. Sequential scanning was performed every 0.2 μm for the reconstruction of the x-z and y-z sections. For immunostaining and the following experiments, including dietary tests, triglyceride, glucose and trehalose measurements, and lifespan, virgin females and males were measured separately.

The following reagents were obtained from the respective commercial vendors. Recombinant human TGF-β1 from R&D Systems (Minneapolis, MN, USA), losartan from Selleck Chemicals (Houston, TX, USA), dexamethasone from Sigma-Aldrich (St. Louis, MO, USA), ganciclovir from Selleck Chemicals, normal horse serum (RTU Vectastain Universal Elite ABC Kit) from Vector Laboratories (Burlingame, CA, USA), murine monoclonal anti-CMV IE antibody (anti-CMV Immediate Early Antigen Antibody, #LS-C103255) from LSBio (Seattle, WA, USA), a goat anti-mouse antibody (VectaFluor R.T.U. DyLight 488 anti-mouse) from Vector Laboratories, rhodamine phalloidin and Vecta-Stain mounting media (VECTASHIELD) from Invitrogen (Carlsbad, CA, USA), a TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit (Total TGF-β1 ELISA Kit with precoated plates) from BioLegend (San Diego, CA, USA), and a MTS assay kit (CellTiter 96^® AQueous One Solution Cell Proliferation Assay) from Promega (Medison, WI, USA).