Potassium ferrocyanide

In order to qualitatively evaluate the levels of MNP uptake in cells by microscopy, 20,000 cells (BxPC-3) or 30,000 cells (Panc-1) were seeded on 8-well culture chamber slides, subsequently cultured, and incubated with enzymes and MNPs as indicated above. Thereafter, the cells were harvested by washing 3 times with HBSS to remove noninternalized MNP. The cells were fixed for 30 min at room temperature (RT) with 3.7% formaldehyde in HBSS, then washed and stained by the Prussian blue method. Furthermore, 3D spheroids were embedded in 1% agarose and then paraffinized, and 5–10-µm thick sections were sliced and dewaxed 2 times by 10 min incubation in toluene followed by fixation /rehydration in a series of 90%, 70%, and 50% ethanol. Subsequently, the dewaxed 3D spheroid slices and fixed monolayer cells were incubated for 10 min in 10% potassium ferrocyanide and then for 30 min in a mixture of 20% hydrochloric acid and 10% potassium ferrocyanide (both from Sigma Aldrich, Steinheim Germany). Finally, the cytoplasma were counter-stained with Eosin B solution. All slides were mounted with Faramount (Dako, Glostrup, Germany) and cover-slipped, and the cells were imaged on an Olympus BX50 microscope.

Whole organs of Ifi27l2a^-/- and wild type mice were fixed with 0.5% glutaraldehyde in PBS for 15 minutes at room temperature, washed twice with PBS, and then stained overnight at 37°C in staining solution containing 1 mg/ml X-gal (Sigma Aldrich, St. Louis, MO, USA), 1 mM MgCl₂, and 5 mM potassium ferrocyanide (Sigma Aldrich). Cryo-sectioning (10 µm) and subsequent LacZ staining were performed as described previously [58] (link). In brief, sections were fixed in 0,5% (v/v) glutaraldehyde/PBS for 10 minutes at 4°C, washed in 1 mM MgCl₂/PBS and stained over night at 37°C in staining solution containing 1 mg/ml X-gal (Sigma Aldrich, St. Louis, MO, USA), 1 mM MgCl₂, and 5 mM potassium ferrocyanide (Sigma Aldrich). The slides were counterstained with Nuclear Fast Red (Sigma) for 1 minute at room temperature.