antibody against cd68 / inos / arg-1 sc-20060  (Santa Cruz Biotechnology)

Journal: Biomaterials Research

Article Title: Decellularized Extracellular Matrix Scaffold Loaded with Regulatory T Cell-Conditioned Medium Induces M2 Macrophage Polarization

doi: 10.34133/bmr.0196

Figure Lengend Snippet: Gene expression regulation in BMDM by dECM within an inflammatory microenvironment. (A) Representative CD68 immunofluorescence staining in BMDMs. (B) Venn diagram of differentially expressed genes within each group. (C) RT-qPCR results detecting the relative mRNA expression within each group. (D to G) Volcano plot (D), heatmap (E), GO terms (F), and KEGG terms (G) of the differentially expressed genes within LPS + IFN-γ and LPS + IFN-γ + dECM groups. In each experiment, n ≥ 3 biological replicates. Bars indicate the mean ± SEM. ns = not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: The following antibodies were used: anti-iNOS (ab178945; Abcam, UK), CD206 (AB64693; Abcam, UK), and CD68 (sc-20060; Santa Cruz Biotechnology).

Techniques: Gene Expression, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Molecular Therapy

Article Title: Multiple-omics analysis reveals a dedifferentiation-immune loop in intrahepatic cholangiocarcinoma

doi: 10.1016/j.ymthe.2025.02.019

Figure Lengend Snippet: Macrophages promote malignant cell dedifferentiation and proliferation (A) The heatmap demonstrates the enrichment of signaling pathways in three different malignant subsets based on GSVA score. The color key ranging from blue to red represents the GSVA analysis score, indicating the degree of enrichment from low to high. (B) mIHC was conducted on ICC tumor samples to validate the activation of the Hippo signaling pathway. The staining included CD31 for endothelial cells, CD68 for macrophages, CD3 for T cells, and αSMA for fibroblasts. The scale bar represents 50 μm. (C) A t-SNE plot was employed to identify four distinct cell types within the macrophage population: CD68 + CD11b low C1QA/B + resident macrophages (ResMf), CD68 + CD11b + infiltrating macrophages (InfMf), SPP1 + macrophages (SPP1+ Mf), and CD1c+ DCs. (D) The heatmap illustrates the expression levels of specific markers in each macrophage subpopulation. The color key ranging from gray to green represents the average expression levels from low to high. (E) mIHC analysis was performed on ICC tumor samples to validate the existence of the four macrophage cell types identified earlier. The staining included CD68 and C1QA for resident macrophages, CD68 and CD11b for infiltrating macrophages, SPP1 for SPP1+ macrophages, and CD1c for CD1c+ DCs. The scale bar represents 50 μm. (F) Ligand-target gene interactions between macrophages and epithelial cells were examined. The heatmap displays the regulatory potential, with the color key ranging from gray to green indicating the degree of potential regulation from low to high. (G) t-SNE plots demonstrate the expression levels of SERPINE1 (left) and VEGFA (right) in different malignant cell subgroups based on scRNA-seq. The color key ranging from gray to blue indicates the relative expression levels from low to high. (H) A t-SNE plot was generated to visualize the expression levels of TGFB1, VEGFA, and IL-6 in different macrophage subpopulations based on scRNA-seq. The color key ranging from gray to blue indicates the relative expression levels from low to high. (I) mIHC staining on ICC tumor samples was conducted to validate the findings of the NicheNET analysis. The staining included CD68 to identify macrophages, and the analysis suggested that CD68 might regulate malignant cells through overexpressed TGFB1 and activated YAP1. The scale bar represents 50 μm.

Article Snippet: Following permeabilization, cells were blocked with 2% BSA for 1 h and then labeled with primary antibodies against CD68 (sc-20060, Santa Cruz), LILRB1 (ab170909, Abcam), or LILRB2 (abs139124, Absin) for 1 h. Subsequently, the cells were incubated with the secondary antibodies for 1 h in the dark.

Techniques: Protein-Protein interactions, Activation Assay, Staining, Expressing, Generated

Journal: Molecular Therapy

Article Title: Multiple-omics analysis reveals a dedifferentiation-immune loop in intrahepatic cholangiocarcinoma

doi: 10.1016/j.ymthe.2025.02.019

Figure Lengend Snippet: Malignant cells induce immunosuppressive macrophages (A) A list of immune checkpoints expressed by TAM was set as GOI. Violin plot showed the expressions of seven GOI immune checkpoints in macrophages based on feature genes from four subcluster macrophages. (B) The interaction potential between the ligands on each malignant cell cluster and the GOIs of macrophages were examined. Using a color key ranging from gray to green, the interaction potential was visualized, with green indicating high interaction potential and gray indicating low interaction potential. (C) To evaluate the prognostic significance of immune checkpoint expression in patients with ICC, a Kaplan-Meier survival curve was constructed based on proteomic data from our in-house cohort of 110 patients with ICC. The p values obtained from two-sided log rank tests were used to assess the statistical significance of the survival differences. (D) IHC staining was performed to examine the expression of two immune checkpoints, LILRB1 and LILRB2, in peritumoral tissues (adjacent normal tissues) and ICC tissues. The staining results were visualized using a scale bar, and a magnification of 40 μm was used. (E) Kaplan-Meier survival curve of LILRB1 (left, p = 0.03, hazard ratio, 0.35; 95% confidence interval [CI]:, 0.14–0.91) and LILRB2 (right, p = 0.078, hazard ratio, 0.39; 95% CI, 0.14–1.11) expression in patients with ICC (from our own collection). p values were obtained from two-sided log rank tests. (F) t-SNE plots were generated to visualize the expression levels of LILRB1 and LILRB2 in the tumor TME of ICC. The color key ranged from gray to blue, with blue indicating high expression levels and gray indicating low expression levels. (G) Flow cytometry analysis was performed to confirm the expression of LILRB1 and LILRB2 by TAM in the TME of ICC. The results showed that TAM indeed expressed these immune checkpoints. (H) IF staining was used to further validate the expression of LILRB1 and LILRB2 by TAM in patients' samples. CD68 was used to label macrophages, and a scale bar representing 10 μm was used to visualize the staining results. (I) Western blot assay was conducted to compare the expression of active YAP1, a downstream signaling molecule, in a normal bile duct epithelial cell line (HIBEC) and several ICC cell lines. For the statistical analysis of active YAP1/actin level, ∗∗∗∗ p < 0.0001. (J and K) IF showed the LILRB1/2 staining. In brief, 80 nM PMA was used to induce THP-1 cell to M0 macrophages and incubated for 48 h then directly co-cultured with normal bile duct epithelial cell line HIBECs and ICC cell line CCLP1 and HCCC9810 for another 24 h. Scale bar, 10 μm ∗∗∗ p < 0.001. All experiments were independently repeated more than three times to ensure reproducibility.

Article Snippet: Following permeabilization, cells were blocked with 2% BSA for 1 h and then labeled with primary antibodies against CD68 (sc-20060, Santa Cruz), LILRB1 (ab170909, Abcam), or LILRB2 (abs139124, Absin) for 1 h. Subsequently, the cells were incubated with the secondary antibodies for 1 h in the dark.

Techniques: Expressing, Construct, Immunohistochemistry, Staining, Generated, Flow Cytometry, Western Blot, Incubation, Cell Culture