Gdf15

For central administration
directly into the CNS, GRASP, and GDF15 were infused in 1 μL
volumes into the fourth ventricle (4th ICV). All systemic treatments
were delivered by intraperitoneal (IP) injection. For central administration,
GDF15 (human recombinant, Biovision, cat. 4569) was dissolved at a
concentration of 30 pmol/μL in 100% dimethyl sulfoxide (DMSO).
For systemic treatments, GDF15 was dissolved in a 5 mM acetate salt,
240 mM propylene glycol, and 0.007% polysorbate 20, in a pH 4 saline
solution and injected at a dose of 20 μg/kg (1 mL/kg). For central
administration, GRASP was dissolved in artificial cerebrospinal fluid
(aCSF; Harvard Apparatus) and injected at 300 and 3000 pmol concentrations.
Systemically delivered GRASP was dissolved in 0.9% saline and injected
in a volume of 1 mL/kg at 30 and 100 nmol/kg. Cisplatin (Cis, cis-diammineplatinum dichloride, Sigma-Aldrich) was dissolved
in 0.9% saline and administered at a dose of 6 mg/kg. The selective
5-HT₃R antagonist Ondansetron (Tocris) was dissolved in
0.9% saline and IP was injected at 2 mg/kg (1 mL/kg). GDF15, GRASP,
and/or Ondansetron were administered IP at 1 mL/kg, and cisplatin
was administered IP at 6 mL/kg. Fluorescently tagged GDF15 and GRASP
were dissolved in aCSF (300 pmol/μL); 1 μL was infused
into the lateral ventricle (LV ICV).

Tissue samples were fixed with 10% formalin for 24 h. Then samples were dehydrated, transparent, waxed and embedded. Paraffin‐coated tissue blocks were sliced into 4‐ to 6‐μm sections. Paraffin sections were deparaffinized in xylene twice for 10 min each time and rehydrated with different concentrations EtOH (absolute ethyl alcohol, 95% EtOH, 80% EtOH) for 5 min each time. After deparaffinization and rehydration, the tissue sections were blocked with 3% H₂O₂ for 10 min. Antigen retrieval was performed with 0.01 m citrate buffer solution (pH 6.0) for 15 min. Then goat serum was added to tissues sections to seal the nonspecific binding site for 15 min. Tissues sections were incubated with the primary antibody against GDF15 (1 : 400; Abcam, Cambridge, MA, USA) at 4 °C overnight and rewarmed at 37 °C for 45 min in the next day. Subsequently, the sections were incubated with horseradish peroxidase‐labeled secondary antibody at room temperature for 20 min. Next, after the addition of diaminobenzidine for color development, the sections were counterstained using hematoxylin. Finally, the images were obtained under a microscope (Olympus, Tokyo, Japan).

The IHC and IFC staining were performed as previously described [29 (link), 30 (link)]. The antibodies used for immunostaining were GDF15 (1:100, Abcam, USA) and β-catenin (1:100, Cell Signaling Technology, USA).