Mildform 20n

Morphometric analysis was performed as previously described [20 (link)]. Lung tissues were fixed with Mildform^®20N (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) at constant pressure (25 cm H₂O). Paraffin-embedded section (5 μm thickness) was stained with hematoxylin and eosin. All histopathological analyses were performed in a blinded manner.

Cells were fixed with Mildform 20N (8% formaldehyde; Wako) for 30 min and permeabilized with 0.2% Triton X-100 (nacalaitesque) for 5 min. Fixed cells were blocked with Blocking One solution (nacalaitesque) for 1h. The primary antibodies used here were: rabbit anti-human Nestin (1:200; N-1602; IBL Ltd.), mouse anti-neuron specific βIII-tubulin antibody (1:500; TuJ-1; R&D Systems), mouse monoclonal anti-MAP2 antibody (1:500; Sigma-Aldrich) and anti-mouse β-catenin (1:1000; BD Bioscience, USA). The samples were washed for three times, and incubated with goat anti-mouse IgG F(ab’)2-TRITC (1: 100; Santa Cruz) and anti-rabbit IgG F(ab’)2 Alexa Fluor 555 conjugate (1: 1000; Cell Signaling Technology) at room temperature for 1 h. After three times of washing, the samples were counterstained with 4’-6-diamidino-2-phenylindol (DAPI, Invitrogen), and examined by confocal inverted microscope (Nikon Eclipse Ti, Japan). Fluorescent images of approximately 400 cells in at least 3 different areas were analyzed for Tuj-positive cells. DAPI was used to quantify the total number of cells.

Tissues were fixed overnight in Mildform 20N (Wako, Osaka, Japan), embedded in paraffin, and sectioned at 4 μm thickness. Sections were immersed in sodium citrate (pH 6.0) buffer for antigen retrieval, and subsequently incubated at 4 °C overnight with primary antibodies as follows; SFXN1 (12296-1-AP, 1:400; ProteinTech, Rosemont, IL), Ki-67 (D3B5, 1:400; Cell Signaling Technology, Danvers, MA) and cleaved caspase-3 (5A1E, 1:2000; Cell Signaling Technology). They were probed with anti-rabbit IgG antibody labelled with Histofine Simple Stain MAX-PO (Nichirei Bioscience, Tokyo, Japan) and visualized with diaminobenzidine (Wako). The TUNEL assay was conducted using DeadEnd Colorimetric TUNEL System (Promega, Madison, WI) according to the manufacture’s protocol. Nuclei were stained with hematoxylin. The intensity score of SFXN1 staining was determined in HCC tissues and adjacent liver tissues of each sample, ranging from 0 to 4. Tumor samples with a score of 0 or 1 and with a score of 2, 3 or 4 were categorized into the low and high expression groups, respectively.