Ab16728

We dropped fresh tumor tissue in 4% paraformaldehyde overnight at 4 °C. After being washed with PBS three times for 10 min, the fixed tissue was placed in 20% and 30% sucrose solution to dehydrate and then was embedded in optimal cutting temperature medium (Thermo Scientific). Embedded tissue was sectioned at 20–30 μm cryosections using Leica CM3050S cryostat. Before IHC staining, tissue slices were subjected to heat-mediated antigen retrieval with Tris/EDTA buffer (PH 9.0). Then, tissue slices were blocked in 5% donkey serum for 2 h. Sections were incubated with primary antibody at the following dilutions: goat anti-IBA1 (1:100, Abcam, ab5076), Rabbit anti-MCP1 (1:100, Abcam, ab73680), mouse anti-CD44 (1:100, Abcam, ab16728), rabbit anti-VWF (1:100, Abcam, ab9378), mouse anti-CD3 (1:50, Abcam, ab699), rabbit anti-cleaved Caspase-3 (1:200, Cell Signaling, #9661), rabbit anti-TMEM119 (1:1000, Abcam, ab185333). After incubation overnight at 4 °C with primary antibody, Alexa Fluor 488, 594, or 647 fluorophore-conjugated secondary antibodies (1:500) (Life Technologies), and DAPI (1:500) were added to slices incubation buffer for 2 h. We used Olympus FV3000 confocal microscope for collecting images.