Vectashield dapi aqueous mounting medium

B16F10 tumor-bearing mice were treated with combination immuno-NPs/anti-PD1, immuno-NPs only, anti-PD1 only, or left untreated on day 7 following inoculation. Mice were euthanized on day 11, 4 days following the start of therapy. Immediately following, organs were harvested, fixed in 4% PFA/PBS, dehydrated in 30% sucrose/PBS, and embedded and frozen in optimum cutting temperature medium (OCT medium, Thermo Fisher Scientific). Primary antibodies to mouse anti-PD1 and anti-PDL1 were purchased from BioXCell, while secondary antibodies were purchased from Thermo Fisher Scientific. 7-μm thick frozen sections were sectioned, blocked with goat serum, and stained with 1:50–1:100 primary antibodies overnight at 4°C, followed by staining with 1:50–1:100 secondary antibodies for 1 hr at 25°C. Following staining, sections were mounted with No. 1.5 glass coverslips using Vectashield DAPI aqueous mounting medium (Vector Laboratories). Samples were imaged using a Leica TCS SP8 gated STED confocal microscope (Leica Microsystems).

4T1 tumor-bearing mice were injected with fluorescent nanoparticles and perfused 24 hr later with PBS and PBS containing 4% paraformaldehyde (Alfa Aesar, Haverhill, MA). Following euthanization, organs were harvested, fixed in 4% PFA/PBS, dehydrated in 30% sucrose/PBS, and embedded and frozen in OCT (Fisher Scientific, Hampton, NH). Primary (anti-CD31, anti-CD11c, anti-F4/80, anti-CD49b) and secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA), Abcam (Cambridge, MA), and BioLegend (San Diego, CA). Frozen sections 10 μm in thickness were stained with 1:100–1:150 primary antibodies overnight at 4°C, followed by staining with 1:100–1:150 secondary antibodies for 1 hr at 25°C. Stained sections were mounted with No. 1.5 glass coverslips using Vectashield DAPI aqueous mounting medium (Vector Laboratories, Burlingame, CA) and imaged using a Leica TCS SP8 gated STED confocal microscope (Leica Microsystems, Buffalo Grove, IL).