Nb110 55674

Lung tumor specimens from human and Kras^LA2 mutant mice were placed in 4% (w/v) paraformaldehyde (PFA) overnight at 4°C and processed for paraffin embedding. 3 μm thick paraffin sections were sliced with the microtome (Zeiss Hyrax M 55) and placed on superfrost plus adhesion slides. De-paraffinized sections were subjected to quenching of endogenous peroxidase activity using a mixture of methanol/H₂O₂ for 20 min, followed by antigen retrieval in a de-cloaking chamber. From this step on, the slides were washed with tris-buffered saline with Tween-20 (TBST, 20 mM Tris, 0.8% NaCl, 0.02% Tween-20, pH 7.6 adjusted with HCl) after each incubation with the reagents throughout the procedure. The sections were incubated first with Rodent Block M (Zytomed Systems) for 30 min and then with the primary antibody, i.e., EGFR (Cell Signaling, D38B1 for human, Abcam, ab52894 for mouse), CCR2 (Novus Biologicals, NB110-55674), or IgG control for 1 h. The cuts were then incubated with Rabbit on Rodent AP-Polymer for 30 min, which was followed by Vulcan Fast Red AP substrate solution (both Biocare Medical) incubation for 10-15 min. Sections were counterstained with hematoxylin (Carl Roth) and dehydrated respectively in consecutively grading ethanol and xylene (both Appli-Chem) incubations. Dried slides were mounted in Entellan and visualized with a slide scanner.

A549 and MH-S cells which were grown on coverslips were exposed to ATTO 633-labeled MSNs for 1 h. Afterwards, the cells were washed three times with PBS, then once with NaCl (0.15 M, pH 3.0), and then three times with PBS again. Cells were fixed with 70% ethanol and permeabilized with 0.1% Triton-X. After another PBS wash, cells were incubated with Roti-Block for 1 h at room temperature. Then, A549 cells were stained with EGFR antibody (Abcam, ab52894) whereas MH-S cells were stained with CCR2 antibody (Novus Biologicals, NB110-55674) overnight at 4°C. The following day, the cells were incubated with the Alexa Fluor secondary antibodies for 1 h at room temperature, washed with PBS, incubated with DAPI for 10 min for nuclear staining, and then mounted with fluorescent mounting medium (Dako).