Alexa 594 donkey anti mouse

To generate expression plasmids, the Cln7 pENTR clone was recombined into pAMW. The full-length Rheb ORF was amplified from clone GH10361 and cloned in to pENTR. After sequence verification it was recombined in pAFW. Tagged proteins were immunoprecipitated with Myc-Trap_MA beads or anti-FLAG M2 Affinity gel overnight at 4 °C. For immunofluorescence, cells were fixed 48 h after transfection in 4% formaldehyde in PBS for 10 min then stained as for larval tissue except that Hoechst was added to the diluted secondary antibodies at 1:10,000 to label DNA. Coverslips were mounted in ProLong Gold (Thermo) and viewed on a LSM780 confocal microscope. Primary mouse anti-Flag and goat anti-myc were the same as for western blots but used at 1:500. Secondary antibodies were Alexa-488 donkey anti-goat and Alexa-594 donkey anti-mouse (1:500; Jackson Immuno Research).

Every fifth 50-μm-thick free-floating brain section from the relevant regions (NAc, VTA, MPFC) of DON injected and control injected rats was processed first for c-Fos immunohistochemistry as described earlier, except that the signal was visualized by incubation in fluorescein isothiocyanate-tyramide (FITC tyramide; 1:8000) and 0.001% hydrogen peroxide in Tris-hydrochloride buffer (0.1 M, pH = 8.0) for 6 min instead of DAB. Subsequently, the sections were incubated in mouse anti-tyrosine hydroxylase (TH) antiserum (1:5000; Chemicon, Temecula, California, United States, catalogue number: MAB5280) or mouse anti-parvalbumin antiserum (1:1000; Sigma, St. Louis, Missouri, United States, catalogue number: P3088) for overnight at room temperature, and then in Alexa 594 donkey anti-mouse (1:500, Jackson ImmunoResearch, catalogue number: 715-585-150) for 1 h. Sections were then mounted, dried, and coverslipped with Aqua-Poly/Mount (Polyscience, catalogue number: 18606).

Immunostaining of adult brains and fat bodies was performed as previously described[25 , 50 (link), 51 (link)]. Tissues were dissected in ice-cold PBS. Brains were fixed overnight in 0.8% paraformaldehyde (PFA) in PBS at 4°C. The fixed brains were washed five times in PBS with 0.5% BSA and 0.5% Triton X-100 (PAT), blocked for 1 hour in PAT + 5% NDS, and then incubated overnight at 4°C with the primary antibodies. Following incubation, the brains were washed five times in PAT, re-blocked for 30 min, then incubated in secondary antibody in block for 4 hr at room temperature. Finally, the brains were washed five times in PAT, then mounted on slides in Slow fade gold antifade. Primary antibodies were as follows: rabbit anti-Dilp5 (1:500; this study); Chicken anti-GFP (1:500; Cat# ab13970, RRID:AB_300798); And Mouse anti-Draper (1:50; DSHB 5D14 RRID:AB_2618105). Secondary antibodies from Jackson ImmunoResearch (1:500) include donkey anti-Chicken Alexa 488 (Cat# 703-545-155, RRID: AB_2340375); donkey anti-mouse Alexa 594 (Cat# 715-585-150, RRID:AB_2340854). Lipid droplets were stained with lipidtox (1:500, Thermo Fisher Cat#H34477) overnight at 4C.