M7018

Explants were processed for immunohistochemical analysis as previously described [10 (link),22 (link)]. Briefly, slides were deparaffinized, rehydrated, and subjected to antigen retrieval with sodium citrate. After blocking with Dako protein block (Dako, Mississauga, ON, Canada), the slides were incubated overnight at 4 °C with primary antibodies: anti-mouse BCRP (1:200, BXP-21, ab3380, Millipore, Billerica, MA, USA), anti-mouse HLA-G (1:300, 4H84, Exbio, Burlington, ON, Canada), and anti-mouse CK7 (1:1000; M7018, Dako). In the controls, mouse or rabbit IgG1 (Dako) was added instead of the primary antibody. After incubation, the slides were washed and incubated with the corresponding biotinylated secondary antibody (1:300, 1 h, Dako). Sections were washed in 1× PBS (3 × 10 min) and incubated with streptavidin-HRP (30 min; Dako); immunostaining was detected with the peroxidase substrate kit DAB (Dako). The slides were counterstained with hematoxylin, dehydrated in ascending concentrations of ethanol, and cover slipped with mounting medium. Visualization was undertaken with an Olympus BX61 upright, motorized microscope, and representative images were captured using an Olympus DP72 digital camera (Olympus, Tokyo, Japan).

Following initial tissue preparation, slides were incubated with both mouse anti-cytokeratin (1:50, Dako M7018) and rabbit anti-vimentin (1:250, Abcam ab92547, Cambridge United Kingdom) primary antibodies overnight at 4°C in 1% BSA in PBS. PBS-T washes were then performed before incubation with goat anti-mouse IgG H&L Alexa Fluor^® 594 (1:500, Abcam ab150116) and goat anti-rabbit IgG H&L Alexa Fluor^® 488 (1:500, Abcam ab150077) secondary antibodies, for 1 h at RT in 1% BSA in PBS. PBS-T washes were performed before applying DAPI-ProLong Gold Antifade solution (Invitrogen) and cover slips were applied. Finally, slides were imaged at 20x magnification using an Axio Imager M2 microscope (Zeiss) and analysed using Zen imaging (Zeiss) and ImageJ software (Fuji) (Abràmoff et al., 2004 ).

The immunohistochemical stain for CK7 (1 : 200, Dako M7018, mouse monoclonal, clone OV-TL 12/20), CK19 (1 : 50, Dako M0888, mouse monoclonal, clone RCK108), and CD34 (1 : 160, Dako M7165, mouse monoclonal, clone QBEnd 10) was performed on the 5-micrometer thick tissue sections of paraffin-embedded tissue blocks. Antigen retrieval was carried out with 0.01 M citrate buffer at pH 6.0. The slides were stained on the Dako Autostainer (Dako Corporation, Carpinteria, CA).
Sections of various control tissues (breast for CK7, liver for CK19, and colon for CD34) with known positivity for the target proteins were used as positive stain controls. Positive staining was defined as dark brown staining pattern. Scant or fine granular background staining or no staining at all was considered negative.

The cells were fixed with 4% paraformaldehyde (PFA; Sigma) for 30 min at room temperature and washed with phosphate-buffered saline (PBS) three times. Then, the cells were permeabilized with 0.2% TritonX-100 (Sigma) for 20 min at room temperature and washed with PBS three times. The cells were blocked in 3% BSA (Applichem) and 5% donkey serum (Merck) in PBS for 2 h at RT. Immuno-labeling was performed with CDX2 (EPR2764Y, ABCAM; 1:250), KRT7 (M7018, DAKO; 1:100), OCT3/4 (sc-5279, SantaCruz; 1:100), NANOG (AB21624, ABCAM; 1:100), and mouse IgG (0.4 μg/mL) overnight at +4 °C. Next day, the cells were washed with 1X PBS three times and they were incubated with appropriate secondary antibodies conjugated with Alexa-Flour 488, 555, or 568 for 3 h at +4 °C in dark. The cells were washed again with 1X PBS for three times. Images were acquired using a confocal microscope (Leica TCS SP8, USA) and processed via ImageJ.