Hematoxylin

To measure intracellular lipid accumulation, H9c2 cells were stained by Oil Red O dye (Sigma-Aldrich, catalog number 398039) according to the methodology of previous study [23 (link)]. After treatment, cells were washed and fixed by 4% (v/v) paraformaldehyde. Then the cells were incubated with the Oil Red O working solution for 30 min at room temperature. Subsequently, the cells were washed twice with 60% isopropanol and then counterstained with hematoxylin (Dako, USA) for 30 s. Excess hematoxylin was washed in water. Cells were then observed under the microscope and images were collected using an ECLIPSE 50i system (Nikon, Japan). Oil Red O-staining positive cell counts were determined over five viewing fields and averaged. Totally more than 150 cells in each group were chosen randomly for statistical analysis.

Fixed tissue was processed using an autoprocessor machine (Leica, Wetzlar, Germany) via dehydration with increasing concentrations of ethanol, followed by clearing with xylene, and then embedded in pure paraffin wax (Sigma, #327204). Then, 5-μm sections of FFPE tissue sections were deparaffinized in xylene (Sigma, #534056) for 10 min, hydrated in decreasing concentrations of alcohol (Merck, Burlington, MA, USA, #100983), and then immersed in tap water. For hematoxylin/eosin staining (H&E), sections were stained in hematoxylin (Dako, Glostrup, Denmark, #S3309) for 30 s, washed with tap water (alkaline medium), and then stained with eosin (Dako, #CS701) for 1 min. For collagen I and III staining, slides were incubated in picrosirius red stain (Abcam, Cambridge, UK, #ab150681) for 1 h then washed in 0.5% acetic acid for differentiation. Stained sections were dehydrated with incremental concentrations of ethanol, cleared with two changes of xylene (Sigma, #534056), and then mounted with DPX (Sigma, #06522). Mounted sections were scanned with a slide scanner (Zeiss, Oberkochen, Germany) and analyzed using Zeiss blue software.

PCa cells seeded on coverslips in 6-well plates were treated with Etn for different time points. Cells were fixed in 10% formalin for 30 min, followed by 5 min incubation with 60% isopropanol. LDs were stained with Oil Red-O staining (ORO) dye (Sigma-Aldrich, #O0625), and cell nuclei were counterstained with hematoxylin (Agilent Technologies, #CS700). Coverslips were mounted with Aquatex (Millipore, #108562) and imaged under a light microscope equipped with a Fein Optic 3DCxM12 camera. Densitometry analysis was performed using ten images per time point. Images were color-thresholded using ImageJ to select for the stained areas. The percentage of stained areas was calculated as follows: % ORO = (red area) / (total occupied area) × 100.

The extraorbital lacrimal glands were removed from euthanized mice and fixed in 10% neutral formaldehyde for 18–24 h. The preparations were washed with phosphate-buffered saline and then subjected to increasing ethanol concentrations from 30%, 50%, 70%, 90%, and then 100% 3 times. Each ethanol treatment lasted for 8–12 h to ensure thorough dehydration. The samples were processed through xylenes and embedded in Surgipath Paraplast (Leica, Deer Park, IL, USA, cat no. 39601006). Tissue sections were cut at 5 μm thickness and dried at 60 °C. The sections were then processed for Hematoxylin–Eosin staining through dewaxation, rehydration, and staining in Mayer’s Hematoxylin (Agilent Technologies, Santa Clara, CA, USA, cat. no. S3309) for 90 s, followed by being washed 3 times and being de-stained with acidic alcohol for 10 s. After being stained with 3% Eosin Y (Sigma-Aldrich, St. Louis, MO, USA, cat. no. E6003), the slides were mounted by coverslips with Micromount (Leica, Deer Park, IL, USA, cat. no. 3801731).

A DeadEnd™ Colorimetric TUNEL System (Promega, Madison, WI, USA) was used for the assay. Liver slides were treated with xylene and ethanol, NaCl, 4% paraformaldehyde, and proteinase K, followed by treatment with rTdT at 37 °C for 2 h. Hydrogen peroxide, HRP, DAB, and hematoxylin (Agilent) were added to the slides, which were incubated for 30 min and then treated with ethanol and xylene. The slides were visualized at 200× magnification using a K1-Fluo microscope (Nanoscope Systems).

Serial 4-µm-thick sections were cut from each 10% formalin-fixed and paraffin-embedded primary tumor specimen collected by pleural biopsy or radical surgery, then evaluated using immunohistochemistry staining according to standard protocols. Sections were heated in 0.01 M citrate buffer (pH 6.0; cat. no. RM102-C; LSI Medience Corporation) at 98°C for 15 min for antigen retrieval and incubated in 3% hydrogen peroxide (cat. no. 081-04215; FUJIFILM Wako Pure Chemical Corporation) for 10 min to inactivate endogenous peroxidase. After blocking with Protein Block Serum-Free (cat. no. X090930-2; Agilent Technologies, Inc.) for 15 min, sections were incubated with mouse anti-podoplanin monoclonal antibody (clone D2-40; cat. no. 413451; pre-diluted antibody; Nichirei Biosciences, Inc.) and rabbit anti-EGFR monoclonal antibody (1:50 dilution; clone D38B1; cat. no. 4267S; Cell Signaling Technology, Inc.) for 1 h. Sections were then washed and incubated with Histofine Simple Stain MAX PO (MULTI) (cat. no. 424152; Nichirei Biosciences, Inc.) for 30 min. Thereafter, sections were visualized with DAB+ Liquid (cat. no. K346811; Agilent Technologies, Inc.) for 10 min and counterstained with hematoxylin for 1 min (cat. no. 30002; Muto Pure Chemicals Co., Ltd.). All steps after the antigen retrieval step were performed at room temperature.