A 2.1 kg, spayed female, 10-year-old Yorkshire Terrier dog presented to a local hospital with dyspnoea. The presence of pericardial fluid was confirmed on ultrasound, which was then collected under ultrasound guidance, and the physical and chemical properties of the pericardial fluid were examined to determine its characteristics. The bacterial and fungal culture tests yielded negative results, indicating no growth. There were no abnormalities in complete blood count and serum chemistry. The patient was referred to Kangwon National University Veterinary Teaching Hospital. Total 30 ml of pericardial fluid was collected from the area where it was observed under ultrasound guidance with local anaesthesia; 1 mg/kg (0.2 ml) of alfaxalone was intravenously administered, followed by an additional 0.1 ml. After 10–15 min, 4 ml of 2% lidocaine diluted in saline (1:1) was administered. Bloody pericardial effusion was smeared and cytological examination was performed. In cytological examination, binucleation, anisocytosis, anisokaryosis, abnormal nucleoli (large, angular, and multiple), abundant basophilic cytoplasm, high nuclear–cytoplasmic ratio (N:C), and coarse chromatin, and large atypical multinucleate cells, indicating high-grade malignancy were observed. These cells can be found either as individual or as clumps of aggregated with a number of erythrocytes. Numerous non-degenerative neutrophils and macrophages were observed alongside these cells. Erythrophagia was observed, indicating chronic hemorrhage. Distinguishing between reactive mesothelial cells and mesothelioma in the pericardial fluid is challenging, especially in the presence of neutrophil-rich inflammation. Therefore, the author aimed to discover whether the observed cells were reactive mesothelial, mesothelioma, or adenocarcinoma cells via immunocytochemistry using five markers (cytokeratin, vimentin, desmin, E-cadherin, and calretinin) [, –]. Immunocytochemistry was performed by modifying the method recommended by the antibody manufacturers; mesothelioma cells as positive control for each of the primary antibodies were prepared from stored smear cells, which were collected from a dog patient diagnosed as mesothelioma post-mortem. At that time, the mesothelioma cells were smeared and fixed in methanol for 10 min and stored in a glass jar containing 1x phosphate-buffered saline (PBS) at 4 ℃ for several months. For calretinin and E-cadherin staining, cell smears from pericardial effusions were fixed in methanol at -20 °C for 10 min and washed thrice with PBS (Sigma-Aldrich, Burlington, MA, USA) for 2 min. The primary antibodies against calretinin (1:1000, Sigma C7479; host: rabbit; reactivity: human, mouse, dog) and anti-E-cadherin, Alexa Fluor 594 (10 µg/ml, Biolegend 147,306; host: rat; verified reactivity: mouse, human; reported reactivity: cynomolgus, dog, pig), were diluted with 1% bovine serum albumin (BSA)/PBS (Komabiotech, Seoul, South Korea). Following overnight incubation with the primary antibodies at 4 °C, the slides were rinsed thrice in PBS for 2 min. The secondary antibody Alexa Fluor 488 (10 µg/ml, Invitrogen, A11034) goat anti-rabbit immunoglobulin G (IgG; heavy + light chain [H + L]) was diluted with 1% BSA/PBS. Following overnight incubation with the secondary antibodies at 4 °C, the slides were rinsed thrice in PBS for 2 min. The slides were then counterstained with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) and examined using an LSM 780 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany). For cytokeratin and vimentin staining, pan cytokeratin monoclonal antibody (AE1/AE3), eFluor™ 570 (1 µg/ml, Invitrogen 41-9003-82, Carlsbad, CA, USA), vimentin monoclonal antibody (V9), and fluorescein (1 µg/ml, Invitrogen 11-9897-82) were used; the same protocol (staining method or time required) as described above was performed using a different secondary antibody. For desmin staining, desmin monoclonal antibody (D33) (1:100, Invitrogen MA5-13259) was used as a primary antibody, and Alexa Fluor 488 (1:500, Invitrogen A28175) goat anti-mouse IgG (H + L) was used as a secondary antibody. The same procedures as those described above were performed. Immunocytochemistry of pericardial fluid was positive for vimentin and cytokeratin. DAPI and the merged image of Fig. A–C are presented in Fig. D. It was also positive for desmin. DAPI and the merged image of Fig. E, F are shown in Fig. G, H. Finally, it was positive for calretinin and E-cadherin. DAPI and the merged image of Fig. A–C are shown in Fig. D. Therefore, malignant mesothelioma was diagnosed [,, –].