Eosin solution

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 micro-CT analysis, each of the three samples were rinsed in PBS, embedded in Tissue-Tek ® O.C.T. freezing compound (Sakura Finetek, Netherlands), then stored at −80°C. 14 μm thick histology specimens were cut from frozen block using a motorized Microm HM 550 cryostat (Thermo Fisher Scientific) and placed onto glass slides. Samples were H&E stained using Gill II Hematoxylin solution (Leica, Wetzlar, Germany) and with a Eosin solution made from Eosin Y powder (Fisher Scientific, Pittsburg, PA), and were Alizarin Red stained with a solution made from Alizarin Red S powder (Sigma-Aldrich), and Von Kossa stained using a kit (ab150687, Abcam, Cambridge, UK), then imaged using a NanoZoomer Digital Pathology System (Hamamatsu, Japan). All images taken were analyzed qualitatively.

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).