Bluing solution

At the termination of treatments, the mice for each group were sacrificed, and the harvested organs were fixed using 4% paraformaldehyde at 4 °C overnight, then immersed in 70% ethanol, embedded in paraffin, and sliced at 5 μm thickness using a Leica cryo-microtome (RM2255, Leica, GE). These sections were stained in hematoxylin (Sigma-Aldrich) for 2 min, rinsed with water, and transformed in 1% HCl acid/alcohol for 30 s. Then the slices were washed and immersed in bluing solution (Thermo Fisher Scientific) for 1 min, washed with water and rinsed in 10 drops of 95% ethanol. The slides were then counterstained in eosin by dipping into ethanol diluted eosin (Thermo Fisher Scientific) solution (ethanol:eosin = 1:5) for a few seconds, dehydrated using 95% alcohol, then xylene for 5 min, each. Finally, the slides were mounted with xylene-based mounting medium (Permount, Sigma-Aldrich) and imaged using a Nanozoomer system. (Hamamatsu, Japan).

After completing the study, we fixed the excised tissues in 4% paraformaldehyde overnight at 4 °C, immersed them in 70% ethanol, embedded them in paraffin, and then sliced at 5 μm thickness using a Leica cryo-microtome (Leica RM2255). We stained these sections in undiluted hematoxylin (Sigma-Aldrich, USA) for 2 min, rinsed in running water, and differentiated in 1% HCl acid/alcohol for 30 s. We then washed these and immersed them in bluing solution (Fisher, USA) for 1 min, washed in running water and rinsed in 10 dips of 95% alcohol. After this, we counterstained the slides in eosin by dipping into 1:5 ethanol diluted eosin solution (Fisher) for less than 30 s, dehydrated through 95% alcohol, and then xylene for 5 min each. We mounted slides with xylene based mounting medium (Permount, Sigma-Aldrich, USA) and imaged using Nanozoomer (Hamamatsu, Japan).

After 24 h of nanoparticle administration tumor tissues were excised by lobectomy and processed for ex vivo histological analysis. Excised tissues were fixed in 4% paraformaldehyde overnight at 4 °C and immersed in 70% ethanol, and then embedded in paraffin, sliced at 5 μm thickness in a Leica microtome (Leica RM2255). Sections were stained in undiluted hematoxylin (Sigma-Aldrich, USA) for 2 min, rinsed in running water, and differentiated in 1% HCl acid/alcohol for 30 s. They were then washed and immersed in bluing solution (Fisher, USA) for 1 min, washed in running water and rinsed in 10 dips of 95% alcohol. After this, slides were counterstained in eosin by dipping into 1:5 ethanol diluted eosin solution (Fisher) for a total of less than 30 s, dehydrated through 95% alcohol, absolute alcohol, and xylene for 5 min each. Slides were mounted with xylene based mounting medium (Permount, Sigma-Aldrich, USA) and imaged using a Nanozoomer (Hamamatsu, Japan).