*2.3. Immunohistochemistry*

Sections from each block were cut at 4 μm and stained using commercially available antibodies (SDHA Mouse Monoclonal Antibody [2E3GC12FB2AE2, ThermoFisher Scientific, catalog #459200] at 1:100 dilution and SDHB Rabbit Polyclonal Antibody [ThermoFisher Scientific, catalog #PA5-23079] at 1:75 dilution). Staining was performed on a Leica Bond immunostainer per the manufacturer's directions. Sections from 20 canine adrenal glands and 20 cardiac samples were used to verify the appropriate immunoreactivity of the antibody with canine antigens.

Pheochromocytoma samples from dogs were listed as positive when there was a granular intracytoplasmic immunoreactivity with a similar intensity as the internal positive controls (endothelial

cells, sustentacular cells, and/or lymphocytes). Negative samples lacked immunoreactivity in the cells of the mass that showed immunoreactivity in internal positive controls [9].

## *2.4. Statistical Analysis*

Descriptive statistics were performed using JMP Pro 12 software (SAS Institute Inc., Cary, NC, USA) 21. Descriptive statistics that were calculated included averages and percentiles. The frequencies of positive SDHA and SDHB immunoreactivity between di fferent variables (invasion, sex, breed, age, and animal weight) were evaluated and compared using Chi-square tests. For all statistical analyses, *p* > 0.05 was considered significant.
