Sample Selection
Feline hepatocellular carcinoma specimens were selected from archival histology libraries at Tufts University, Cummings School of Veterinary Medicine and Louisiana State University (LSU) School of Veterinary Medicine, Louisiana Animal Disease Diagnostic Laboratory (LADDL). Seven cases were identified. Additionally, 7 sex (3 male, 4 female) and age-matched (8 to 14 years), control cases with normal hepatic histology were selected from the LADDL archives. The formalin-fixed, paraffin-embedded liver specimens were sectioned and stained with hematoxylin & eosin (H&E). Slides were reviewed for diagnostic criteria by a veterinary anatomic pathologist (IML). Matched controls samples were deemed free of steatosis and inflammatory cells, which could influence hepatocellular ploidy.
Immunofluorescence staining protocol: Five µm sections were cut and mounted on charged slides. Samples were deparaffinized in xylene and serially rehydrated using a descending gradient of ethanol-water solutions. Slides were washed in phosphate buffered saline with 0.1% Triton X-100 (PBST). After citrate buffered antigen retrieval, tissues were blocked with 5% normal goat serum at room temperature for one hour. Tissues were incubated with β-catenin primary antibody (1:200, Invitrogen Beta-catenin polyclonal antibody; Carlsbad, CA) in 1% in normal goat serum at room temperature for 2 hours, washed with PBST, and incubated with secondary antibody (Biotium CF594 F(ab’) 1:1000; Fremont, CA) for 1 hour at room temperature. Hoechst 33342 (H42) nuclear stain (1ug/mL, Thermo Scientific) was applied for 20 minutes at room temperature. After dye incubation, slides were washed for 5 minutes with PBS. Slides were cover slipped using the Biotium EverBrite Hardset Mounting Medium (Fremont, CA) and allowed to dry for a minimum of 30 minutes before microscopic analysis.
Microscopic Analysis: Modeled after Toyoda et al. 2005, slides were examined using integrated fluorescence microscopy (Zeiss AXIO Observer Z1, Carl Zeiss, Göttingen, Germany). The Zeiss Neofluar 40X/0.75 EC plan objective (Fluorescence, high transmission) was used for all images acquired. The Zeiss #64 filter set was used to image β-catenin (Excitation:587/25 and Emission: 647/70). Hoechst nuclear imaging was performed using Zeiss #34 filter set (Excitation 390/22 and Emission 460/50). To ensure that the same cells were not counted twice, slides were read in a systematic manner, moving from top right to left of the slide and then on successive descending lines. Each captured image contained approximately 25 square mm of surface area. Digital images were collected and merged with Zen2 software (Zen Pro).
Ploidy measurement
Quantification of cellular ploidy (mono- or binucleate) was enabled by β-catenin immunofluorescent staining to outline the plasma membrane. Nuclear ploidy (chromosome number per nucleus) was quantified using H42 staining. H42 stoichiometrically binds to the minor groove of DNA when crosslinking fixatives are used. A minimum of 100 cells per section were analyzed. Cells were excluded if they displayed overlapping nuclei or indeterminate plasma membrane borders.
Statistical Analysis
A diploid feline cell contains 38 chromosomes (19 pairs), differing from mice and humans, thus a species specific intensity distribution was generated. The individual nuclear intensities werecombined in multinucleated cells to represent the total cellular ploidy. Measurements from histologically normal feline liver were compiled and graphed based on intensity per nuclei and probability density. The cutoff intensity for cell ploidy was determined by fitting two Gaussian mixture with “fitgmist” package from MATLAB ver R2020a. All other analyses were performed with JMP Pro 15 (SAS Institute Inc., Cary, NC). The intensity and ploidy distributions within each cell, among three different cell types, were compared using Kolmogorov-Smirnov test (Biesterfeld et al. 1994). P < .05 was considered significant.