Dpx mounting medium

Liver tumor organoids were fixed in 4% paraformaldehyde (Tech and Innovation, Chuncheon, Republic of Korea) for 1 h and incubated overnight at 4 °C in 30% sucrose phosphate buffer. Thereafter, the liver organoids were embedded in FSC-22 frozen section medium (Leica Biosystems, Wetzlar, Germany) and cut into 10 µm-thick sections using a cryostat at −20 °C. The sections were then permeabilized using 1% Triton X100 in phosphate-buffered saline (PBS) for 10 min, and blocked using blocking buffer (5% bovine serum albumin in PBS containing 0.2% Triton X100) for 30 min. The sections were then incubated overnight at 4 °C with primary antibodies against CK8 (sc-8020; Santa Cruz Biotechnology), EMR1 (sc-377009; Santa Cruz Biotechnology), and cleaved caspase-3 (CC3, MAB835; R&D Systems) in blocking buffer. For fluorescence labeling, the sections were incubated with Alexa Fluor 488- or Alexa Fluor Plus 647-conjugated secondary antibody (1:100; Invitrogen, Carlsbad, CA, USA) for 1 h at 25 °C. Nuclear staining was conducted using DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA), and samples were mounted using DPX Mounting Medium (Electron Microscopy Sciences, Hatfield, PA, USA). The sections were then observed and imaged under a confocal microscope (LSM800; Zeiss, Oberkochen, Germany).

Brain sections were mounted on slides and dried overnight under vacuum. Slides were washed (2×10 min) in KPBS, and then fixed for 20 min in 4% (w/v) PFA. They were then dehydrated according to the following sequence: H₂O, 50% (v/v) EtOH, 70% (v/v) EtOH, twice in 95% (v/v) EtOH, and thrice in 100% (v/v) EtOH; each dip carried out for 3 min), transferred next into xylene for 5, 30 and 2 min, and finally rehydrated using the reverse-order sequence (thrice in 100% (v/v) EtOH, twice in 95% (v/v) EtOH, and then 70% (v/v) EtOH, 50% (v/v) EtOH and H₂O; 2 min for each dip). Slides were next dipped 20 times in 0.25% thionin (Sigma-Aldrich). Mounted brain slides were dehydrated again (20 dips in each of H₂O, 50% (v/v) EtOH, and 70% (v/v) EtOH, and then 2 and 3 cycles of 3-min dips in 95% (v/v) and 100% (v/v) EtOH, respectively, and finally, 2 cycles of 3-min dips in xylene) before overlaying with coverslips in DPX mounting medium (Electron Microscopy Sciences).
For stereological analysis, 3 sections (−1.46, −2.06, and −2.46 mm from the bregma) were analyzed using the Stereo Investigator software. For each section, the cornu ammonis 1 through 3 (CA1/CA2, CA3) areas and the dentate gyrus area were defined and expressed as ratio of total hippocampus area for both hemispheres. Photographs were obtained with a Nikon C80i microscope equipped with a QImaging® color camera.