4 6 diamidino 2 phenylindoldihydrochloride dapi

Cytospin preparations of cultured PBMCs and tumor-derived cells were completed by washing cells twice with phosphate buffered saline (PBS; Life Technologies, Carlsbad, CA), adjusting the cell suspensions to a concentration of 2 × 10⁵ cells/ml, and centrifuging 50 μl of cell suspension at 550 rpm for 1 min using a cytocentrifuge (Thermo Fisher Scientific, Waltham, MA). Cytospins were incubated with the primary or isotype control antibody followed by incubation with the corresponding FITC-conjugated secondary antibody. Cytospins were subsequently incubated with the second primary or isotope control antibody followed by incubation with the corresponding cyanine 3 (Cy3)-conjugated secondary antibody. Cytospins were counterstained with 4′,6-diamidino-2-phenylindoldihydrochloride (DAPI; Sigma-Aldrich, Steinheim, Germany), covered with polyvinyl-alcohol mounting medium [1,4-diazabicyclo[2.2.2]octane (DABCO); Sigma-Aldrich], and analyzed using a Zeiss microscope (Zeiss, Oberkochen, Germany). The quantification of each immunofluorescent staining was performed by two independent investigators blinded for the underlying disease. The chosen magnified fields were representative for the whole cytospin.

Cells were fixed in either 100% ice-cold methanol or 4% paraformaldehyde in PBS for 15 min at room temperature, washed three times with PBS (Sigma), and blocked for 30 min with either 10 or 40% goat serum (Sigma) in PBS. Cells were incubated with primary antibody diluted in 10% or 40% goat serum overnight at 4°C on a rocking platform (see Additional file 1: Table S1). Cells were washed three times with PBS and incubated with goat anti-mouse or anti-rabbit Alexa Fluor 488 (1:200; Life Technologies) in 10 or 40% goat serum. For co-staining of nestin and VE-cadherin, mouse isotype-specific antibodies were used (1:200; Life Technologies). Cell nuclei were labeled with 4′,6-Diamidino-2-pheny-lindoldihydrochloride (DAPI; Sigma) for 10 min. Cells were washed three times with PBS and visualized.

Micropatterned tissues were rinsed with PBS and fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) in PBS for 10 min at room temperature. Following three rinses with PBS, the micropatterned substrates were blocked and permeabilized in PBS with 5% Donkey Serum and 0.3% Trition X-100 (TX100; Thermo Fisher) (PBS-DT) for 1 hr at room temperature. Next, the tissues were incubated with primary antibodies diluted in PBS-DT for 2 hr at room temperature or overnight at 4°C. Then, the substrates were rinsed in PBD-DT three times for 15 min each. Secondary antibodies were diluted in PBS-DT and exposed to the tissues for 1 hr at room temperature. A list of primary antibodies and dilutions can be found in Table S3. Tissue nuclei were subsequently stained with 300 nM 4’,6-diamidino-2-pheny-lindoldihydrochloride (DAPI, Sigma Aldrich) for 30 min followed by three 15 min PBS washes. The micropatterned substrates were mounted on glass coverslips using Prolong Gold Antifade Reagent (Thermo Fisher). Brightfield images of micropatterned tissues were obtained using a Nikon TS100 microscope with a ME600L camera. Fluorescence images were obtained using a Nikon A1R confocal microscope.