Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: ZBP1 is mainly expressed in macrophages and alters the ratio of M1 and M2 macrophages in sepsis‐induced myocardial dysfunction. A single‐nucleus mRNA sequencing (snRNA‐seq) was performed in hearts from LPS‐treated WT or Zbp1 −/− mice (for each group, single‐cell suspensions from three hearts were pooled as one sample). (A) UMAP plot of cell clusters in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. CM, cardiomyocyte; EC, endothelial cell; FB, fibroblast; Mac, macrophage; NP, neutrophil. (B,C) UMAP plot and dot plot of Zbp1 expression in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. (D,E) UMAP plot of macrophage in myocardial tissues from WT and Zbp1 −/− mice induced by LPS. M1, M1 macrophage; M2, M2 macrophage. (F–H) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images (F) and quantification of positive cells (G,H) are shown [scale bar = 50 µm, n = 5]. Mean ± SEM; * P < 0.05, *** P < 0.001, ns, no significance. WT, wild‐type; ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; snRNA‐seq, single‐nucleus mRNA sequencing.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Sequencing, Expressing, Marker, Staining, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Myeloid‐specific Zbp1 deficiency protects against sepsis‐induced myocardial dysfunction. (A) Schematic showing experimental design to investigate the impact of myeloid‐specific Zbp1 deficiency on septic myocardial dysfunction (LPS: 10 mg/kg; 6 h). (B) Zbp1 gene knockout efficiency in hearts and bone marrow cells was detected by Western blot. (C–E) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in Zbp1 fl/fl or Zbp1 cko mice. M‐mode analysis of EF (D) and FS (E) before (pre) and 6 h after (post) LPS treatment. [scale bar = 20 µm, n = 7 in each group]. (F–H) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 7 in each group). (I,J) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. LPS, lipopolysaccharide; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Gene Knockout, Western Blot, Staining, Immunohistochemistry, Injection, Marker

Journal: Clinical and Translational Medicine

Article Title: Myeloid deficiency of Z‐DNA binding protein 1 restricts septic cardiomyopathy via promoting macrophage polarisation towards the M2‐subtype

doi: 10.1002/ctm2.70315

Figure Lengend Snippet: Blocking STAT1 prevents cardiac dysfunction caused by sepsis. (A) Schematic showing experimental design to investigate the impact of STAT1 inhibition on septic myocardial dysfunction (Flu, fludarabine, STAT1 inhibitor, 50 mg/kg/day; Sol, solvent). (B–D) Western blotting detection and quantitative analysis of STAT1 and ZBP1 in myocardial tissues at 6 h after intraperitoneal injection of NS or LPS (10 mg/kg) in solvent or fludarabine‐treated mice ( n = 5 in each group). (E–G) Representative images of transthoracic M‐mode echocardiographic trance, hematoxylin and eosin (H&E) staining, immunohistochemistry staining of CD11b, Ly6C and Ly6G at 6 h after intraperitoneal injection of LPS in solvent or fludarabine‐treated mice. M‐mode analysis of EF (F) and FS (G) before (pre) and 6 h after (post) LPS treatment. [Scale bar = 20 µm, n = 8–9 in each group]. (H–J) Quantitative analysis of CD11b + , Ly6C + area and Ly6G + cells in myocardial tissues ( n = 5 in each group). (k–l) Heart tissues were labelled with M1 macrophage marker iNOS and M2 macrophage marker CD206. Tissues were counterstained with DAPI. Representative staining images and quantification of positive cells are shown [scale bar = 50 µm, n = 5 in each group]. Mean ± SEM; * P < 0.05, *** P < 0.001. ZBP1, Z‐DNA binding protein 1; LPS, lipopolysaccharide; STAT1, signal transducer and activator of transcription 1; EF, ejection fraction; FS, fractional shortening; DAPI, 4′,6‐diamidino‐2‐phenylindole.

Article Snippet: For intracellular marker CD206 (E‐AB‐F1135H, Elabscience) staining, the cells were fixed with a fixation buffer and permeabilised with a permeabilisation buffer before incubation with the fluorochrome‐conjugated antibody against CD206.

Techniques: Blocking Assay, Inhibition, Solvent, Western Blot, Injection, Staining, Immunohistochemistry, Marker, Binding Assay

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet: Combination of CITE-seq, scRNA-seq, snRNA-seq, and spatial analyses enables identification of all hepatic cell types including bona fide cell doublets, related to Figure 1 (A and B) Top DEGs (A) and DEPs (B) for cell types from Figure 1 B. (C) Distinct profiles of cells or nuclei within the UMAP depending on isolation protocols; 71,162 cells from ex vivo digestions, 96,066 cells from in vivo digestions, and 18,666 nuclei. Numbers on plots represent numbers of cells/nuclei per population. (D) Correlation plots showing genes captured within the KC, B cell and neutrophil populations with and without addition of CITE-seq antibodies. (E) Expression of VSIG4, CD206, and ESAM (protein, top) and Vsig4 , Mrc1 , and Esam (mRNA, bottom). (F) UMAP showing clusters of cells when only minimal QC for gene number and % mitochondrial genes is performed; 17,669 cells pooled from 3 samples. Expression of Cd5l, Cd19 , and Kdr by the clusters facilitating identification of cell types per annotation. (G) CITE-seq data from (F) in Flow-Jo showing expression of CD206 and ESAM in total KCs (left) and total B cells (middle). Numbers represent % of entire KC or B cell population. Identified populations were then mapped back onto the original UMAP (right). (H) Expression of CD31, CD26, and CD38 by indicated populations. (I) Heatmaps showing expression of top DEGs between KC1s and LSECs (left), KC2s and KC1s + LSECs (middle) and B cell2s and B cell1s + LSECs (right). (J) 3D reconstruction of murine liver following perfusion with antigen fix to inflate endothelial cells and staining with antibodies against CD31, CD206, and F4/80. (K) UMAP showing clusters generated from Visium analysis of liver tissue (4 samples) and liver capsule (1 sample). (L) Top unbiased genes defining zonation trajectory from portal to central vein in Visium. (M) Expression of Glul and Epcam by confocal microscopy (left), annotation of portal, periportal, mid, and central regions on same tissue section (middle) and overlay of both datasets (right). (N) Identification of cholangiocyte (left) and cDC (right) signatures on zonated Visium spots. (P) Molecular Cartography showing expression of indicated zonated hepatocyte mRNAs in liver tissue. Data are representative of 2 mice. (O) Expression of Itgae (encoding CD103) in the UMAP of the total liver (left) and flow cytometric analysis of total cDC1s for CD103 and MHCII expression in the healthy murine liver (right).

Article Snippet: Anti-Human CD206 (REAL518) APC , Miltenyi Biotec , 130-122-168; RRID: AB_2857557.

Techniques: Isolation, Ex Vivo, In Vivo, Expressing, Staining, Generated, Confocal Microscopy

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet:

Article Snippet: Anti-Human CD206 (REAL518) APC , Miltenyi Biotec , 130-122-168; RRID: AB_2857557.

Techniques: Purification, Recombinant, Staining, cDNA Synthesis, Gene Expression, Software, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Fraxin Attenuates Rheumatoid Arthritis by Regulating Macrophage Polarization and Inhibiting Fibroblast-like Synoviocyte Proliferation

doi: 10.3390/ijms27072946

Figure Lengend Snippet: Fraxin inhibited inflammation in Raw264.7 cells and FLs. ( A , B ) Effect of Fraxin on the cellular viability of Raw264.7 and FLs. ( C ) EC-50 of Fraxin (100 ng/mL) in Raw264.7 cells. ( D – F ) Flow cytometry analysis showing the polarization effect of Fraxin (100 ng/mL) on Raw264.7 cells. ( G – L ) RT-PCR analysis of Il6 , Tnfα , Il1b , Il10 , Cd206 , and Hspa8 in FLs treated with Fraxin. ( M – O ) Protein levels of p-NFκB-p65 and TNF-α were examined by Western blot in FLs. Fraxin low-dose group (Fraxin-L, 100 ng/mL), Fraxin high-dose group (Fraxin-H, 1 μg/mL). ## p < 0.01, ### p < 0.001 versus Ctrl; ** p < 0.01, *** p < 0.01 versus LPS.

Article Snippet: CD206 (A02285-2) and CD86 (BM4121) were purchased from Boster Biological Technology Co., LTD (Wuhan, China).

Techniques: Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Fraxin Attenuates Rheumatoid Arthritis by Regulating Macrophage Polarization and Inhibiting Fibroblast-like Synoviocyte Proliferation

doi: 10.3390/ijms27072946

Figure Lengend Snippet: Fraxin improved the inflammatory microenvironment of the knee joint. ( A ) H&E staining of the knee joint. The area within the box represents the magnified region shown below. The red arrow points to meniscal injury, the blue arrow points to synovial thickening, and the green arrow points to synovial inflammatory pannus. ( B ) Knee joint staining with saffron solid green. ( C , D ) CD206 and CD86 immunofluorescence staining in the synovium. ( E ) The pathology score from H&E staining. ( F , G ) Quantitative analysis of CD206 and CD86 immunofluorescence staining. ( H ) Serum levels of IL-6 were analyzed by ELISA. ( I ) Serum levels of TNF-α were analyzed by ELISA. ## p < 0.01 versus Ctrl; ** p < 0.01 versus Model.

Article Snippet: CD206 (A02285-2) and CD86 (BM4121) were purchased from Boster Biological Technology Co., LTD (Wuhan, China).

Techniques: Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay