0.1 m sodium citrate

Sagittal sections were performed at 4 μm thickness from paraffin blocks of enucleated eyes from in vivo tumor models (n = 6) and human patients (n = 6). Immunohistochemical analysis with human eyes was approved by Institutional Review Board of Seoul National University Hospital (H-1404-032-568) and clinical demographics of 6 patients were summarized in Supplemental Table 1. We also followed Declaration of Helsinki during the whole procedures with human tissue samples. The sections were incubated at 60°C for 2 hours and processed with deparaffinization and hydrated by sequential immersion in Xylene Substitute (Thermo) and graded ethyl alcohol solutions. Antigen retrieval was performed by the treatment with 0.1 M sodium citrate (pH 6.8, Sigma) at 120°C for 10 minutes. The sections were permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes. Then, we treated the sections with 1X Universal Blocking Reagent (Biogenex) for 10 minutes to minimize nonspecific binding. After incubation with primary antibodies (1:250) overnight, the sections were treated with REAL™ Detection Systems (Dako) and DAB Kit (Life Technologies) as the manufacturer's instructions. Then, the slides were mounted with Permount solution (Thermo) and observed under the light microscope (Nikon).

Intracellular accumulation of lipids was determined by using Oil-Red-O (ORO) (Sigma Aldrich, Madrid, Spain) which binds to neutral lipids. Before staining, preparations were washed once with PBS followed by 60% isopropanol. Staining was carried out at 37 °C for 5 min, then washing was performed with 60% isopropanol and PBS, and finally preparations were visualized using Leica DFC 7000T microscope (Leica Microsystems, Wetzlar, Germany). For quantification, ORO staining was extracted with 1 mL 100% isopropanol and the optical density of the samples was measured at a wavelength of 510 nm using a microplate reader Infinite 200 PRO spectrophotometer (Tecan, Männedorf, Switzerland). Cells were also stained with crystal violet solution and dye was extracted for quantification with 50% 0.1 M sodium citrate (Sigma Aldrich, Madrid, Spain) and 50% ethanol and absorbance was measured at 590 nm. Alternatively, organoids-derived cells were stained with hematoxylin, washed twice with distilled water, and, finally, visualized using Leica DFC 7000T microscope (Leica Microsystems). For quantification, randomly selected fields were captured and lipid content was quantified using IP Win32 software v4.5.0.29.

Male and female mice aged between 12–18 weeks old were used for these studies. Gast p59−79, ChgA 435−462a and Sst 25−36 were administered intravenously (i.v.) at 1 mg/kg and subcutaneously (s.c.) at various doses (see Fig. 7A, B and C for details on specific doses for each peptide). Blood samples were collected using EDTA coated microvette tubes (Starsedt, Leicester, UK) from the tail vein at various time points over 90 min. Blood was centrifuged (2000 g, 10 min, 4⁰C) and the plasma separated. A 0.1 M sodium citrate (Sigma-Aldrich) buffer (adjusted to pH 3 with citric acid (Sigma-Aldrich)) was added to the plasma at a dilution of 1 in 10 to prevent degradation of Gast p59−79 and Sst 25−36. Plasma was stored -70⁰C till extraction.