Dab substrate

The expression of Lin28A and Lin28B at protein level in colon cancer tissues was detected by immunohistochemistry as previously described [6 ]. Briefly, 5-μm-thick colon cancer tissue sections were prepared. After deparaffinization, rehydration and blocking endogenous peroxidase, the antigen was retrieved by microwave treatment. The sections were blocked by using 5% bovine serum albumin and then incubated with primary antibodies against Lin28B (Abcam, Cambridge, MA, USA) or Lin28A (Abcam) at 4°C overnight. After incubation with secondary antibody labeled with streptavidin-biotin peroxidase for 1h at room temperature, DAB substrate (ZSGB Bio, Beijing, China) was applied for staining.
The result was evaluated by a pathologist who was blinded to the clinical information. The staining score was given by allying intensity with extent. Staining intensity was quantified as follows: negative (0), weak (1), moderate (2), or strong (3). Staining extent was scored according to the percentage of positive cells: none (0), <25% (1), 25-50% (2), 50-75% (3), or >75% (4). The final score was calculated as, the intensity score × the extent score.

Our experimental aim was to investigate the mechanism by which tumor cells modulate immunity during blood metastasis. Hence, we built a blood metastatic model by injecting scramble or mimic 1E8 cells into the right atrium of mice (n = 12, 6 each group) after anesthesia. Animals were handled according to the guidelines and protocols supported by the Animal Care and Use Committees (supplementary animal ethnic).The important tissues of these mice were fixed with 4% paraformaldehyde for histological examination or immunohistochemistry. Hematoxylin‐eosin (H&E) staining was performed by a series of steps, including gradient dehydration, xylene clearing, wax embedding, dewaxing, and staining. For immunohistochemistry, tissue sections were routinely dewaxed, subjected to antigen retrieval and hydrogen peroxide incubation, blocked with goat serum for 0.5 hours, and incubated with the primary antibody overnight at 4°C. The primary antibodies were as follows: CD68 mouse monoclonal antibody (66231‐2‐Ig, Proteintech, 1:2000) and PSAT1 polyclonal antibody (A14124, ABclonal, 1:500). The secondary antibody (enzyme‐labeled goat anti‐mouse/rabbit IgG polymer, PV‐6000, ZSGB‐BIO) was incubated the next day for 0.5 hours. Finally, DAB substrate (ZLI‐9017, ZSGB‐BIO) was added to the sections for chromogenic analysis. The pathologic results were judged by an experienced pathologist.

Mice were intraperitoneally injected with either proteins or PBS at 36 h intervals for 12 weeks, then sacrificed 2 h after the last of these injections. The aortic roots were embedded in OCT (SAKURA, USA) and quickly frozen to −20 °C; then 8-μm serial sections of the aortic root were collected for atherosclerosis analysis. Aortas fixed in 4 % paraformaldehyde were analyzed en face. The extent of atherosclerosis was determined by Oil Red O staining and quantification with Image J software as described previously [32 (link)].
The location of MsrA in the aortic root was detected by immunofluorescence staining with anti-MsrA antibody. Rat anti-mouse monocytes/macrophage antibody-2 (MOMA2, Bio-rad, USA) was used for macrophage detection in the aortic root. Immunoreactivity was visualized using the Vectastain ABC kit (Vector Labs, USA) and then reacted with DAB substrate (ZSGB-BIO, China). Analysis of apoptosis in aortic root tissue was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nickend-labeling assay kit (TUNEL, Roche Diagnostics, Germany) according to the manufacturer’s instructions. The TUNEL positive cells were counted and normalized to the cell totals. MOMA-2 staining area and apoptosis were analyzed with Image-Pro Plus 6.0.

Tissue slides of tumors were prepared from samples collected from in vivo experiments. H&E staining was performed as described previously [33 (link)].
To detect Ki-67-expressing cells, immunohistochemistry was performed on tissue slides. Ki-67 antibodies were purchased from ZSGB-BIO (Cat# ZM-0166, Beijing, China) and Affinity Biosciences (Cat# AF0198, Cincinnati, OH, USA). Second antibody was peroxidase-conjugated goat anti-rabbit IgG (ZSGB-BIO, Cat# PV-6000). DAB substrate (ZSGB-BIO) was used for visualization of positive cells.

Tissue slides were prepared from tumors collected from xenograft experiments. H&E staining was performed as previously described [19 (link)].
Immunohistochemistry was performed to detect expression of Ki-67 and cleaved caspase-3 in tumor tissues. Anti-Ki-67 and peroxidase-conjugated goat anti-rabbit IgG were from ZSGB-BIO (Beijing, China). Anti-cleaved caspase-3 (Asp175) was from Cell Signaling Technology. DAB substrate was used for visualization (ZSGB-BIO).

For immunohistochemical (IHC) staining, formaldehyde-fixed, paraffin-embedded sections of tumor tissues were obtained from mouse models. Briefly, the deparaffinized sections were then treated with methanol containing 3% hydrogen peroxide for 15 min. The sections were washed with PBS and blocked with blocking serum for 30 min at room temperature. The sections were then incubated with primary antibodies, including anti-SMURF2 (bs-4056R, Bioss), anti-ID2 (MA5-32891, Invitrogen), and anti-p21 (ab107099, Abcam) at 4°C overnight. The following day, the primary antibodies were washed off and sections were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The nuclei were stained with hematoxylin, and DAB substrate (ZLI-9019, ZSGB-Bio, Beijing, China) was added to detect the proteins. The obtained immunohistochemistry images were analyzed with following scoring criteria. Cells with 0% staining were scored as 0; cells with 1%-33% staining were scored as 1; cells with 34%-66% staining were scored as 2; cells with 67%-100% staining were scored as 3. Additionally, the staining intensities were evaluated into four grades: 3 (strong), 2 (moderate), 1 (weak) and 0 (none). The final score was defined as the score of percentage classifications multiplied by intensity grades.