The present experiments were conducted in accordance with the Guidelines for Veterinary Clinical Research of Kagoshima University in Japan (No. KVH190001). All samples were surgically removed, fixed in formalin, and subjected to histopathological analysis by veterinary pathologists with no prior knowledge of the cytological results. Histopathological analysis revealed a diagnosis of epithelial or mesenchymal tumor in all cases.
The following criteria were used to select random tissue samples from dogs hospitalized at Kagoshima University Veterinary Teaching Hospital in Japan: 1) clear histopathological diagnosis of epithelial or mesenchymal tumors, 2) cytological diagnosis corresponding to the histopathological diagnosis, and 3) a large number of epithelial or mesenchymal tumor cells with adequate cytomorphology on smear slides. Exclusion criteria were as follows: 1) clear histopathological diagnosis of non-neoplastic masses, round cell tumors, or tumors of unknown origin; 2) multiple populations of neoplastic cells in histological and cytological tissues; and 3) poor cytomorphological quality in cytological samples.
According to these criteria, smear slides were prepared from neoplastic tissues removed from 16 dogs. Smears obtained from 14 dogs were completely dried in air, following which they were cryopreserved at − 30°C within 24 hours after sampling. The duration of storage varied from 2 months to 4 years and 8 months. Prior to ICC, slides were removed from the freezer and immediately and thoroughly dried with a hair drier using the cold setting. The remaining two samples (one epithelial tumor case and one mesenchymal tumor case) were intentionally left at room temperature (RT) for 1 week in a non-air-conditioned room.
ICC for cytokeratin and vimentin was performed in accordance with a previously described method (Sawa et al. 2017). The primary antibodies included mouse monoclonal anti-cytokeratin (clone AE1/AE3, ready‐to‐use; Dakocytomation, Glostrup, Germany) and mouse monoclonal anti‐vimentin antibodies (clone V9, 1:100 dilution; Thermo Fisher Scientific, Fremont, CA, USA). These primary antibodies have been confirmed to be cross-reactive for canine tissues (Fant et al. 2004; Sawa et al. 2012; Ramos-Vara et al. 2016). Biotin‐labeled horse anti‐mouse IgG (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody. The samples were then treated with peroxidase-labeled streptavidin (KPL, Gaithersburg, MD, USA), and all antibodies that did not include anti-cytokeratin were diluted in a mixture of 0.25% casein/10 mM phosphate‐buffered saline (PBS). This same casein/PBS mixture was used as the blocking solution. Reactivity was evaluated using 3,3′‐diaminobenzidine (DAB) chromogen (DAB‐buffer tablet; Merck, Darmstadt, Germany) using normal mouse IgG (Dakocytomation) as a negative control. For ICC, the smear slides were completely dried using the cold setting of a hair dryer. Following fixation with cold acetone for 1 min, slides were washed with PBS for 10 s, blocked with 0.25% casein/PBS for 10 min at RT, and incubated with the primary antibody for 10 min at 37°C. After sufficient washing with PBS, the slides were incubated with secondary antibody for 10 min at 37°C and washed with PBS, following which they were incubated with peroxidase-conjugated streptavidin for 10 min at 37°C. After washing with PBS, samples were stained with DAB for 5 min to examine immunoreactivity. The reaction was terminated with cold distilled H2O, and slides were counterstained with Carrazi’s hematoxylin.
The primary and secondary antibodies mentioned above were also used for IHC, which was performed using the same methods as ICC, except that the antibodies for cytokeratin and vimentin were incubated for 20 min at RT, the secondary antibody was incubated for 30 min at RT, and peroxidase-conjugated streptavidin was incubated for 30 min. Immunoreactivity was visualized using DAB with normal mouse IgG (Dakocytomation) as a negative control.
In the present study, all smears and sections were evaluated as positive (+) or negative (-) for immunoreactivity using light microscopy. Signal strengths were compared between ICC samples and negative controls. The ICC signals were also compared with the standard IHC signals.