Axio imager m2 microscopy system

Eight µm-thick tumor cryosections were fixed with 80% methanol (v/v) aqueous solution for 5 min and afterwards with -20 °C cold acetone for 2 min. Immunofluorescent stainings were performed with the following primary monoclonal antibodies: rat anti-mouse CD31 (PECAM-1) antibody (BD Biosciences, Germany); biotin anti-mouse smooth muscle actin (SMA) antibody (Progen Biotechnik, Heidelberg, Germany); rat anti-mouse F4-80 antibody (AbD Serotec, Puchheim, Germany); and rat anti-mouse CD206/Mrc-1 antibody (Acris Antibodies, Herford, Germany). Secondary antibodies were obtained from Dianova (Hamburg, Germany). DAPI (Sigma Aldrich, Taufkirchen, Germany) was used for nuclear counterstaining and sections were embedded with Mowiol® 4-88 (Roth, Karlsruhe). Images were acquired using the Axio Imager M2 microscopy system (Carl Zeiss AG, Jena, Germany).

Fluorescence microscopy (Axio Imager M2 microscopy system, Carl Zeiss, Göttingen, Germany) was applied to DAPI stained cryosections (n=5 images per section) from tumor, liver and spleen. Afterwards, tissue sections were fixed with methanol 80% and -20°C acetone, and washed three times with PBS (Life Technology, Germany). Via the fixation and washing steps, extracellularly localized micelles were removed. Fluorescence images were captured at exactly identical locations. The signal of the micelles was quantified using AxioVision SE64 Rel. 4.8 software (Carl Zeiss, Jena, Germany) and compared between both pre- and post-washing images. The data obtained as percentage of area fraction was plotted as average ± standard deviation in GraphPad Prism 8 (GraphPad Software, USA).