Ab237723

The liver, lungs, and tumors were immediately fixed in 4% neutral-buffered formaldehyde or Bouin’s solution (Sigma-Aldrich). The tissue was then dehydrated in a gradient of ethanol concentrations and embedded in paraffin. Sections of 5 µm were stained with hematoxylin/eosin or Gomori trichrome to visualize small tumors and metastasizing B16F10 cells. For immunohistochemical staining of CD8+ T lymphocytes and NK cells within tumors, the formaldehyde-fixed samples were processed through a saccharose gradient, embedded in a freezing medium (Tissue Freezing Medium, Leica Biosystems), frozen at -80°C and cut into 10 µm sections on a Leica CM 1860 UV cryotome. Afterward, antigen retrieval was performed on the sections, which were boiled in a sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a microwave oven at 750 W for 6 minutes. The sections were then incubated overnight at 4°C with either anti-CD8 alpha antibody diluted 1:200 (Abcam, ab237723) or anti-NK1.1 antibody diluted 1:100 (Abcam, ab289542) and visualized by fluorescently-labelled secondary antibodies. The slides were then mounted with 15 µl of Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories), upon which they were examined and photographed using a fluorescent microscope (Olympus BX51 with an Olympus DP-2 camera).

Tissue sections (5-μm thick) were placed on a slide and dried in the incubator at 37°C. After xylene dewaxing, the slices were immersed in 50%, 70%, and 80% ethanol for 2 min each and in eosin for 5 s. The specimens were then routinely dehydrated, made transparent, and fixed. The sections were washed with water, the antigen was heat repaired, the primary antibody was incubated, and the secondary IgG was incubated with appropriate biotin (1:1,000, #S0001, Affinity Biosciences, Wuhan, China). The slices were stained with hematoxylin for 5 min, rinsed with tap water for 3 min, rinsed with 1% hydrochloric acid for 2 s, rinsed with tap water for 2 min, and then sealed by gradient dehydration. For IF staining, the specimens were covered with a fluorescent-labeled antibody and stored in an enamel box for 30 min. Examination of co-stained sections and images were collected using a light microscope (Olympus, Tokyo, Japan). The corresponding primary antibodies were against CD80 (ab254579.html">ab254579, Abcam, Cambridge, UK), CD86 (ab254579.html">ab119857, Abcam), F4/80 (ab60343, Abcam), iNOS (ab178945, Abcam), CD4 (ab183685, Abcam), CD8 (ab237723, Abcam), CD3 (ab135372, Abcam), CD44 (ab243894, Abcam), and Il-12R (ab282729, Abcam).