*2.1. Protocols and Ethics Statement*

All experiments were performed on group-housed *N. furzeri* belonging to the long-lived strain MZM 04/10 (Leibniz Institute on Aging Friz-Lipmann Institute, Germany, Jena) at the following time points: 5 weeks post hatching (wph) (young-adult) and 27 wph (onset of aging-related features). Animal maintenance was performed as described [24]. Animals were bred and kept in FLI's fish facility according to paragraph 11 of the German Animal Welfare Act. The protocols of animal maintenance were approved by the local authority in the State of Thuringia (Veterinaer- und Lebensmittelueberwachungsamt) with license number J-003798. Euthanasia and organ harvesting was performed according to paragraph 4 (3) of the German Animal Welfare Act and The Council of The European Union Directive of 22nd of September 2010 (2010/63/UE).

#### *2.2. Animals and Tissue Preparation*

Fish at the selected time point were euthanized at 10 a.m. with an overdose of anesthetics. Fish, without prior sedation, were placed in a buffered Tricaine methanesulfonate solution (MS-222, TricanePharmaq, Pharmaq) at a concentration of 1 mg/mL for approximately 5–10 min until no vital signs were observed (body and operculum movement, righting reflex), followed by decapitation. The whole heads, brains, and intestines were dissected and processed according to the experimental protocols. For RNA extraction, brains were immediately processed as described in Baumgart et al. 2014 [33]. For morphological analysis, the whole heads were opened by a small incision to allow penetration of a fixative and were fixed in paraformaldehyde (PFA, 4% in diethylpyrocarbonate treated phosphate saline buffer (PBS)) overnight (ON) at 4 ◦C and the brains were prepared the next day to maintain structural integrity. For cryostatic embedding, tissues were successively incubated in 20% and 30% sucrose solution ON at 4 ◦C, embedded in cryomount (Tissue-Tek® O.C.T.™, Sakura Finetek USA Inc., Torrance, CA, USA), and frozen at −80 ◦C. Serial coronal sections of 14 μm thickness for the brain and sagittal sections of 16 μm for the intestine were cut with a Leica cryostat (Deerfield, IL, USA). For paraffin embedding, tissues were dehydrated in a graded ethanol series, embedded in paraffin, and serial coronal 7 μm thick sections were cut at the microtome.
