Envision rabbit kit

IHC was performed on FFPE tissue from patient’s primary tumor samples, the CDX and the cell line using with anti-PSA, AR, CK7, CK8/18, EpCAM, Ki67 antigen, Vimentin, CD44, NSE, Chromogranin A, and Synaptophysin antibodies (Supplementary Table 1). IHC was performed manually using the coverplate system. Two protocols were developed depending of the species used to raise primary antibodies (mouse or rabbit) to avoid background due to mouse cells present in the CDX. Antigen retrieval was performed by pH8 EDTA buffer incubation for 40 or 60 min at 98 °C (Supplementary Table 1). Mouse Klear kit (GBI labs) and EnVision rabbit kit (Agilent Technologies, Dako) were used for primary mouse and rabbit antibodies, respectively. Primary antibodies were incubated at RT for 1 h. IHC stains were examined by an experienced pathologist (J.Y.S.).

Lung sections were stained using an envision+ rabbit kit (Agilent-Dako UK), mouse anti-Ki67 (Clone MiB1, Agilent-Dako UK) and rabbit anti-α-smooth muscle actin antibody (ab5694, Abcam UK). Antigen retrieval was achieved by pre-treatment with citrate at pH 6 and 110 °C for 5 min. Sections were then incubated with the primary antibody (1:400 dilution) overnight at 4 °C.

For porcine tissue samples, immunohistochemical staining against human MHC class I was performed (antibody: mouse anti Human HLA Class I Heavy Chain, Clone—HC10—Cat. Nr. MUB2037P) on dewaxed paraffin embedded slides using the DAKO rabbit envision kit, according to manual’s instructions. The antibody was used in a 1:200 dilution. Slides were counterstained in Hematoxylin and Eosin (H&E), dehydrated and mounted in Pertex before photographs were taken at 20× and 40× magnification, using light microscopy.