Pbs buffered formalin

For the immunocytochemistry, cells were fixed in 10% PBS buffered formalin (VWR) and left to incubate at 4 °C overnight before being cut into 3 μm sections and placed on glass slides. After being deparaffinized and rehydrated, sections were exposed to induced antigen retrieval to unmask epitopes. Sections were boiled for 10 min in Envision Flex Target Retrieval Solution, high pH (Dako, Glostrup, Denmark), diluted 1:50 in miliQ H₂O, before being incubated for 1 h with primary ABCB1 antibody (Abcam) diluted 1:1000 in antibody diluent with background reducing components (Dako). Then, sections were washed twice in TBS + 0.5% Triton X-100 and incubated for 20 min with a High Definition Polymer Detector (AH diagnostics, Tilst, Denmark). Colorimetric signals were detected using DAB. Sections were developed with EnvisionTM FLEX DAB + Chromogen (Dako) diluted in EnvisionTM FLEX Substrate Buffer to visualize the primary antibody. Sections were counterstained with Mayer’s hematoxylin (Hounisen, Skanderborg, Denmark) and mounted with Pertex xylene-based mounting media (Hounisen).

Fresh frozen tissue slides were air dried, blocked with BB and incubated with BaxB01 (4 μg/ml, in PDAB) for oxMIF detection. The specimen was fixed in 10% PBS buffered formalin (VWR). Furthermore, the sections were incubated with monoclonal mouse anti-Cytokeratin 20 (Dako M7019, 1:50) or anti-CD31 antibodies (e-bioscience 13-0319, 1:200), and monoclonal rabbit anti-BaxB01 antibodies (0.5 μg/ml) diluted in PADB+0.25% TritonX-100. Rabbit and mouse antibodies were detected by Alexa Fluor^® conjugated secondary antibodies (Life Technologies A11034 and A21424, 1:2000) diluted in PADB+0.25% TritonX-100. The slides were rinsed in PBS and coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Life Technologies). Pictures were taken at 20-fold magnification (LUCPlanFLN 20x, NA 0.45) with an Olympus inverted microscope (VS81) using a mercury lamp and the filters DAPI (U-MWU2), FITC (U-MWIBA3) and TRITC (U-MWIGA3), and a XM10 camera (Olympus).