Assessment of CXCR4, CXCL12, HER2, ER, PR and Ki-67 tissue status by immunohistochemistry (IHC)

IHC was conducted as previously described [18, 24]. Briefly, a representative area of each tumor lesion with a diameter of 6 mm and 3 μm thickness were mounted on Superfrost® plus microscope slides (ThermoFisher Scientific, Waltham, USA). Xylene was used to deparaffinize and an ethanol/water gradient series was used to rehydrate the sections. For CXCL12 immunostaining, tissue slides were immersed in Novocastra™ epitope retrieval solution pH 6 (Leica Biosystems, Wetzlar, Germany) for CXCL12 staining and then boiled in a microwave for antigen retrieval (25 min at 600 W). The staining was performed using the Novolink™ Polymer Detection System, Leica Biosystems, following the manufacturer’s procedure. The following primary human antibodies diluted in Lab Vision™ Antibody Diluent OP Quanto (ThermoFisher Scientific) were used: rabbit monoclonal anti-CXCR4 antibody (clone UMB2, 1:500, Abcam, Cambridge, UK), mouse monoclonal anti-CXCL12α antibody (clone 79,018, 1:50, R&D Systems, Minneapolis, USA), mouse anti-HER2 (clone CB11, 1:200, Invitrogen, Carlsbad, CA, USA), mouse anti-ER (clone 6F11, 1:125, ThermoFisher Scientific), rabbit anti-PR (clone 1E2, ready-to-use, Ventana, Tucson, USA) and rabbit anti-Ki-67 (polyclonal, 1:500, ThermoFisher Scientific). Samples of FMC known to have high CXCR4 and CXCL12 expression and feline tonsil tissue sample were used as positive controls. Tissue sections incubated with no primary antibodies and feline mammary normal samples were used as negative controls. All slides were scored in a blind manner by two independent pathologists and in doubtful and/or divergent IHC results, cases were re-evaluated using a multiobserver microscope and the staining was discussed until a consensus was achieved. Images were taken with an optical microscope system (Axiovert S100 with AxioCam HRc; Carl Zeiss BV, Sliedrecht, the Netherlands) and analyzed using AxioVision (Carl Zeiss).