Roti mount flourcare

Fibroblasts were grown in glass Petri dishes (∅ 5.5 cm) for three days in RPMI1640 medium as described above. Subsequently, the medium was removed and 2 mL PBS buffer was filled into the dishes. Allicin was added to a final concentration of 100 µM, 10 µM, 1 µM or 0.1 µM and cells were incubated at 37 °C and 5% CO₂ for 10 min. As control, water was added in the same volume as allicin solution. Cells were twice washed with PBS buffer and fixed by adding 2 mL of 3.7% formaldehyde (Carl Roth, Karlsruhe, Germany) in PBS at room temperature. Cells were washed again twice again with PBS to remove the formaldehyde. To permeabilize the cells, 2 mL of 0.1% Triton-X 100 (Applichem, Darmstadt, Germany) in PBS were added to the cells for 5 min at room temperature, followed by repeated washing with PBS. To stain the cells with phalloidin-Rhodamine (Aatbioquest, Sunnyvale, CA, USA) in DMSO, the reagent was added according to the manufacturer's instruction and incubated for 20 min in the dark. Nuclei were counterstained using DAPI (1 µg/mL in methanol). After removal of the staining solution and washing, mounting medium was added (Roti®-Mount FlourCare, Carl Roth, Karlsruhe, Germany). Microscopy was performed using a Leica-fluorescence microscope (DM-RBE, Leica GmbH, Wetzlar, Germany), equipped with a rhodamine-filter (Em 590 nm, 20 nm bandwidth).

Frozen tumor samples were cut in 9 μm thick serial sections at −20 °C. Sections were then fixed with cold acetone (≥99%, Fisher scientific, Hampton, VA, USA) for 6 min at −20 °C. To visualize the iron ions, a Perls’ Prussian blue stain was performed. Immunofluorescence staining was used to assess the localization and density of macrophages. The tumor tissue was cut in 9 μm thick serial sections at −20 °C on SuperFrost Plus adhesion slides (Thermo Scientific, Waltham, MA, USA). The sections were incubated overnight at 4 °C with a monoclonal CD68 antibody (1:100) (rat anti-mouse CD68, clone FA-11, Bio-Rad, Hercules, CA, USA) diluted in Dako REAL^TM Antibody Diluent (Dako, Denmark). Following, slides were washed three times with PBS (pH = 7.4). For macrophage visualization, slides were incubated for one hour with AlexaFluor 568 polyclonal secondary antibody diluted 1:200 (goat versus rat IgG, Thermo Fisher Scientific, Massachusetts, USA), then counterstained, and mounted with Roti^®-Mount FlourCare (Carl Roth, Karlsruhe, Germany). In conclusion, the sections were analyzed using a Keyence microscope (BZ-X800 Series, Osaka Prefecture, Japan).

For assessment of LIBS-MB binding in the myocardium, sections from the myocarditis group, which had received LIBS-MB/control-MB, were embedded in Roti®-Mount FlourCare fluorescence medium (Carl Roth GmbH, Karlsruhe, Germany). A fluorescence filter set 38 (BP 470/40, FT 495, BP 525/50) by Zeiss, Germany, was used.
For each mouse, 30 fields of view (FOV) in 40× magnification from 12 different heart sections were evaluated in fluorescence microscopy and the average amount of microbubbles/FOV was calculated.