Superfrost microscopic slides

Cells in suspension were first washed with PBS (LONZA) and cell numbers adjusted to a concentration of 10⁵ ml⁻¹ in PBS. Cells were then adhered to SuperFrost™ microscopic slides (Thermo Scientific) by performing cytospin (Cellspin® II, 3 min at 1500 rpm). Schizonts purified from TBL20 cells were diluted in PBS and attached to poly-L-lysine coated glass cover slips (Corning® BioCoat®). Fixation was done with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA) solution for 10 min at room temperature. Primary antibodies were: rabbit polyclonal antibody against Tamr1 (mAb 1D11), anti-Ta-p104 (Clone IC12, polyclonal mouse antiserum) and rabbit monoclonal anti-acetyl H4 (Sigma-Aldrich). Antibodies were diluted in PBS-1% BSA-0.3% Triton X100 (PBST). Secondary antibodies were a goat anti-mouse Alexa Fluor® 488 and a goat anti-rabbit Alexa Fluor® 594 (Invitrogen™) diluted at 1:2000 in PBS containing 0.3% Triton X100. Cells were finally covered by a round coverslip and sealed using Invitrogen™ ProLong™ Gold antifade mountant with DAPI (ThermoFisher).

Four‐micrometre‐thick tissue sections were cut from selected paraffin‐embedded blocks (Superfrost Microscopic Slides; ThermoFisher Scientific, Bleiswijk, The Netherlands). Slides were deparaffinised, and rehydrated with xylene and ethanol. Endogenous peroxidase was blocked with 0.3% H₂O₂ in phosphate‐buffered saline, and heat‐induced antigen retrieval was accomplished by 15 min of incubation in Tris‐EDTA buffer (pH 9; Klinipath, Duiven, The Netherlands). Mouse monoclonal high molecular weight cytokeratin (clone 34BE12; 1:200; Dako, Heverlee, Belgium) diluted in normal antibody diluent (APG‐500; ScyTek Laboratories, West Logan, WV, USA) was incubated for 2 h at room temperature. Antibody visualisation was performed with the Envision kit (Dako) and slide counterstaining with haematoxylin. When basal cell staining was absent, the cribriform structure was classified as invasive carcinoma; if sporadic, scattered or continuous basal cells were identified, the growth pattern was classified as intraductal carcinoma.

The animals were humanely euthanized after one week and one month post-surgery by administering an overdose of ketamine (100 mg/kg). Tissue samples from the implantation site, liver, and kidneys were fixed in a 10% neutral buffered formalin solution (pH 7.2) for 24 h. For histological analysis, 5 μm thick sections were prepared from paraffin-embedded tissue blocks and mounted on SuperFrost microscopic slides (Thermo Fisher Scientific; Waltham, MA, USA). The slides underwent standard deparaffinization using xylene and rehydration with a series of decreasing ethanol concentrations. Subsequently, the samples were stained with hematoxylin and eosin using conventional methods to enable visual evaluation of the histological specimens. All examinations were performed using a Carl Zeiss Primo Star microscope equipped with a Zeiss AxioCam ERc 5s digital camera and ZEN 2 (blue edition) software (Oberkochen, Germany). Morphometric parameters were assessed in 10 fields of view at ×100 magnification by two independent observers.