Ab213363

LCM was used to isolate CD31+ endothelial cells and CD68+ macrophages from human aortic arch specimens. Briefly, after collection and processing of the aortic arch tissue, which involved fixation, embedding, and sectioning to generate thin tissue slices, the sections were then mounted on the specialized glass slides for LCM and stained with either rabbit anti-CD31 antibody (Ab28364, Abcam, Shanghai, China) for endothelial cells or rabbit anti-CD68 antibody (Ab213363, Abcam) for macrophages, to visualize and identify the target cell populations. Fibronectin staining (Ab2413, Abcam) was performed to help visualization of the tissue. Before performing LCM, the stained tissue sections were dehydrated through a series of alcohol and xylene washes to remove any residual water and to ensure compatibility with the LCM process. The tissue sections were then placed under the LCM microscope, where the regions containing CD31+ endothelial cells or CD68+ macrophages were carefully identified based on fluorescence. Using an ultraviolet laser, the selected regions are microdissected and captured onto a thermoplastic cap to be transferred to a collection tube for protein extraction and ELISA analysis.

Cells were seeded onto glass coverslips in 6-well plates, fixed with 4% formaldehyde in PBS, rinsed with PBS, and permeabilized in 0.5% Triton X-100 for 15 min. Samples were then blocked with 3% bovine serum albumin for 30 min, washed with PBS, and incubated with anti-CD68 antibody (1:200, ab213363; Abcam, Cambridge, MA, USA) or anti-CD163 antibody (1:200, ab87099; Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, samples were washed with PBS and incubated with cyanine 3-conjugated anti-mouse antibody (1:400 dilution; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in the dark for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Immunofluorescence staining of cells was visualized using a laser confocal scanning microscope (Nikon, Tokyo, Japan).

Liver tissue sections were deparaffinized through dimethylbenzene and rehydrated in alcohol gradients. Antigen retrieval was performed using EDTA pH 9.0 buffer at 100 °C for 10 min. Primary antibodies used were as follows: anti-mouse/human CD45 (1:2000, ab208022, Abcam), anti-mouse CD3 (1:500, 17,617–1-AP, Proteintech), anti-human CD3 (1:200, 85061 T, CST), anti-mouse CD19 (1:1000, ab245235, Abcam), anti-mouse F4/80 (1:1000, 28,463–1-AP, Proteintech) and anti-human CD68 (1:5000, ab213363, Abcam). The sections were incubated with primary antibodies overnight at 4 °C. Subsequently, the primary antibodies were detected by incubating the sections with CoraLite488-conjugated secondary antibody (1:500; Proteintech) or CoraLite594-conjugated secondary antibody (1:500; Proteintech) for 1 h at room temperature. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and images were captured using a Zeiss fluorescence microscope.

Tissue slices or VM cells were fixed with 4% paraformaldehyde at 4°C for 15 min and incubated in 0.3% Triton X-100 for 15 min. After blocking with 5% goat serum for 30 min, tissue slices or VM cells were incubated with corresponding primary antibodies against TREM1 (1:200), CD11b (1:200), CD68 (1:200), and VEGFR2 (1:200) at 4°C overnight and then incubated with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated secondary antibodies (Beyotime) for 2 h. DAPI (Beyotime) was used to stain the nuclei. The immunofluorescent signals were detected by fluorescence microscopy (Leica DMi8; Leica Microsystems, Wetzlar, Germany). The following primary antibodies were used: rabbit anti-CD68 (ab213363, Abcam; Cambridge, UK); rabbit anti-VEGFR2 (26415-1-AP, Proteintech Group, Inc.; Wuhan, China).

Following 7 days of co-culture, cancer cell coverslips were removed from the top of the PDMS ring and the monocyte derived cells (MDCs) at the bottom of the microdevice were fixed for 30 min using 4% paraformaldehyde. MDCs were rinsed with 0.1% BSA in PBS then permeabilized and blocked in buffer containing 0.3% Triton X-100 and 1% normal goat serum in PBS for 45 min at room temperature. Cells were then incubated with primary antibodies diluted in dilution buffer containing 1% BSA, 0.3% Triton-X, 1% normal goat serum, and 0.01% sodium azide for 2 h at 25 °C [anti-CD68 (1:200, ab213363, Abcam, Eugene, OR), anti-CD163 (1:100, ab182422, Abcam)]. After rinsing with PBS, all cells were incubated with secondary antibody diluted in the same dilution buffer as the primary antibodies (1:500 goat anti-rabbit IgG Alexa Fluor 488) for 1 h at room temperature protected from light, then counterstained with Hoescht 33,258 for 5 min (1:1000). All images were obtained using a Zeiss Axio Observer.Z1 inverted microscope with an AxioCam 506 mono camera, Plan-Apochromat 20 × 0.16-NA air objective, and Zen 2 software (Zeiss). ImageJ software (NIH) was used to quantify CD68+ and CD163+ counts per field of view, and percent CD68 and CD163 were calculated as the ratio of CD68+ or CD163+ counts to total cell counts from the Hoechst signal.