Journal: Nature neuroscience

Article Title: Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability.

doi: 10.1038/s41593-019-0421-4

Figure Lengend Snippet: Fig. 2 | Cytokine signaling in the LHb for MORwd plasticity. a, Max-projection of confocal image of the LHb with IBA1+ microglia (left). Max-projection of MedLHb microglia in saline + naloxone and NP-MORwd mice (upper right). 3D reconstruction of IBA1+ microglia containing CD68 structures from slices obtained from saline + naloxone and NP-MORwd mice (lower right). b, Analysis of IBA1 microglia immunoreactivity in MedLHb slices (saline + naloxone (n = 4 mice, 366 cells), NP-MORwd (n = 4 mice, 328 cells)). Two-sided t test, t692 = 3.305, ***P = 0.001; 3 independent acquisition sessions. c,d, Quantitative analyses of microglial cell volume (based on IBA1 immunoreactivity) and CD68+ structures. Values are normalized to saline control (saline + naloxone (black; nmice/cells = 4 mice, 89 cells), NP-MORwd (red; n = 4 mice, 83 cells)). IBA1 volume (c): two-sided t test, t170 = 3.05, **P = 0.003. CD68/IBA1 volume (d): two-sided t test, t170 = 2.65, **P = 0.008. Measurements were obtained from independent samples. e, TNF-α (cyan) and DAPI (magenta) immunostaining and normalized TNF-α optical density in the LHb (saline + naloxone (black; n = 7 mice, 6 independent acquisitions per mouse) versus NP-MORwd (red; n = 8 mice, 5 independent acquisitions per mouse)). Two-sided t test, t13 = 2.991, *P = 0.0104. f, Experimental protocol (left) and example traces and AMPAR/NMDAR ratios (right) in the LatLHb with (+) or without (–) exogenous TNF-α (saline + naloxone –TNF-α (n = 2 mice, 9 cells) versus saline + naloxone +TNF-α (n = 2 mice, 9 cells)). Two-sided t test, t14 = 0.37, P = 0.717. g, AMPAR/NMDAR ratios with or without exogenous TNF-α recorded from saline + naloxone (n = 3 mice, 10 cells (–TNF-α) and n = 4 mice, 11 cells (+TNF-α)) and NP-MORwd MedLHb slices (n = 3 mice, 10 cells (–TNF-α) and n = 5 mice, 15 cells (+TNF-α)). Interaction factor F(1,42) = 4.90, two-way ANOVA, *P = 0.039. Data are presented as box plots, with 10th and 90th percentiles, median and scatter.

Article Snippet: PTN5930 CD68 1:400, Bio-Rad Cat. MCA1957, Clone FA-11, Lot.

Techniques: Saline, Control, Immunostaining

Journal: Cancers

Article Title: The Immunological Contribution of a Novel Metabolism-Related Signature to the Prognosis and Anti-Tumor Immunity in Cervical Cancer

doi: 10.3390/cancers14102399

Figure Lengend Snippet: Correlation between the metabolism-related risk score and immune landscape. ( A ) Representative immunostaining pictures of the five hub genes and four cell types (CD4+, CD8+, CD57+ and CD68+ cells). The upper panel comprises images of five hub genes, images of four cell types were in the middle panel. Scale bar: 50 μm. The lower panel illustrates the infiltration scores of tumor-infiltrating CD4+, CD8+, CD57+ and CD68+ cells in the epithelial or stromal cell compartments. * p < 0.05. ( B ) Differences in the infiltration levels of 28 immune cells between high- and low-score groups in the pan-cancer validation cohorts. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in the study were listed as follows: anti-ALOX12B rabbit polyclonal antibody (NBP1-89409, Novus Biologicals, Colorado, CO, USA), anti-FAR2 rabbit polyclonal antibody (NBP1-90435, Novus Biologicals, Colorado, CO, USA), anti-F5 rabbit polyclonal antibody (20963-1-AP, Proteintech, Wuhan, China), anti-CA9 rabbit polyclonal antibody (11071-1-AP, Proteintech, Wuhan, China), anti-TDO2 rabbit polyclonal antibody (15880-1-AP, Proteintech, Wuhan, China), anti-CD4 mouse monoclonal antibody (67786-1-Ig, Proteintech, Wuhan, China), anti-CD8 mouse monoclonal antibody (66868-1-Ig, Proteintech, Wuhan, China), anti-CD57 rabbit polyclonal antibody (19401-1-AP, Proteintech, Wuhan, China) and anti-CD68 mouse monoclonal antibody (66231-2-Ig, Proteintech, Wuhan, China).

Techniques: Immunostaining, Biomarker Discovery

Journal: Regenerative Biomaterials

Article Title: Embedding MSCs in Si-HPMC hydrogel decreased MSC-directed host immune response and increased the regenerative potential of macrophages

doi: 10.1093/rb/rbac022

Figure Lengend Snippet: Analysis of immune innate infiltrate in irradiated colonic mucosa using immunohistochemistry. Representative pictures of ( A ) macrophage infiltrate using CD68 immunostaining and ( B ) neutrophil infiltrate using myeloperoxidase (MPO) immunostaining 7 days after in vivo injection. Positive signal is colored blue. *Indicates the localization of the gel. ( C and D ) Graphs representing the quantification of the blue pixels normalized to a tissue surface located close to the hydrogel and expressed as a percentage. The number of slides analyzed is indicated for each group for CD68 and then MPO immunostaining, respectively. Ir PBS = irradiated and PBS 1×-injected rats ( n = 16/18); Ir gel = irradiated and Si-HPMC-injected rats ( n = 10/6); Ir gel MSC = irradiated and Si-HPMC embedded hu-MSC-injected rats ( n = 16/13).

Article Snippet: For CD68 (macrophages) immunohistochemistry, sections were dewaxed and then treated with proteinase K (DakoCytomation, France) at room temperature (RT) for 5 min, and quenched for endogenous peroxidases by incubation with 3% H 2 O 2 in methanol at RT for 10 min. After saturation, mouse anti-rat CD68 1/200 (AbDserotec, UK) was applied to the section for 1 h at 37°C.

Techniques: Irradiation, Immunohistochemistry, Immunostaining, In Vivo, Injection

Journal: Asian Spine Journal

Article Title: Electron Microprobe Analysis and Tissue Reaction around Titanium Alloy Spinal Implants

doi: 10.4184/asj.2007.1.1.1

Figure Lengend Snippet: ( A ) Metallic debris was identified in the dense connective tissue and the anti Cotrel-Dubousset 68 positive macrophages were observed at tissue adjacent to the metal particles (Avidin-biotin complex, ×100). ( B ) Macrophages as stained positive by anti CD 68 marker (Avidin-biotin complex, ×200, Case 5).

Article Snippet: The macrophages were identified by immunostaining the sections with the anti-CD 68 macrophage marker (Dako) using an indirect immunoperoxidase technique.

Techniques: Avidin-Biotin Assay, Staining, Marker

Journal:

Article Title: The Expression and Distribution of the Hypoxia-Inducible Factors HIF-1? and HIF-2? in Normal Human Tissues, Cancers, and Tumor-Associated Macrophages

doi:

Figure Lengend Snippet: Details of the Primary Antibodies Used

Article Snippet: For some of the CD68 immunostaining biotinylated goat anti-mouse serum at 1/400 (DAKO) was used as the secondary followed by streptABComplex/AP (DAKO).

Techniques:

Journal:

Article Title: The Expression and Distribution of the Hypoxia-Inducible Factors HIF-1? and HIF-2? in Normal Human Tissues, Cancers, and Tumor-Associated Macrophages

doi:

Figure Lengend Snippet: Expression of HIF-2α protein in normal bone marrow macrophages and U937 cell line. Normal bone marrow macrophages show immunoreactivity with mAb 190b. In serial paraffin sections of normal bone marrow trephine staining with CD68 (using mAb PGM1; A), and mAb 190b (B) colocalize in macrophages but not megakaryocytes. U937 cells were cultured with or without PMA (1.6 × 10− 8 M) and incubated in normoxia (N) or 0.1% hypoxia (H) for 4 hours. Whole cell extracts (75 μg) were prepared, separated by SDS-PAGE, transferred onto PVDF membrane, and analyzed with mAb 190b (C). For comparison a hypoxic cell extract of HIF-2α transfected HT1080 cells (50 μg) prepared after culture for 4 hours in 0.1% hypoxia was run in parallel (lane C). The dominant band detected in each case comigrated at the mobility predicted for HIF-2α. In both differentiated and undifferentiated U937 cells the band detected showed hypoxic induction. However, after differentiation the normoxic levels seen were comparable with those seen in hypoxia in undifferentiated cells. U937 cells were cultured with PMA (1.6 × 10− 8 M) to allow differentiation into macrophages, incubated in normoxia or placed in 0.1% hypoxia for 4 hours, and immunostained with mAbs to HIF-2α (190b) and CD11c (KB 90). HIF-2α expression was absent after normoxic culture (D) but detectable after hypoxic culture (E). CD11c expression, confirming macrophage differentiation, was unaffected by normoxic/hypoxic culture (not shown). Original magnifications, ×400 (A and B) and ×300 (D and E)

Article Snippet: For some of the CD68 immunostaining biotinylated goat anti-mouse serum at 1/400 (DAKO) was used as the secondary followed by streptABComplex/AP (DAKO).

Techniques: Expressing, Staining, Cell Culture, Incubation, SDS Page, Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: An Inhibitor of Grp94 Inhibits OxLDL-Induced Autophagy and Apoptosis in VECs and Stabilized Atherosclerotic Plaques

doi: 10.3389/fcvm.2021.757591

Figure Lengend Snippet: HCP1 improved aortic atherosclerotic plaque stabilization in apoE −/− mice. From the top to the bottom panel, masson trichrome staining of collagen (in blue) (A) , immunostaining for mouse α-smooth muscle actin (B) , in situ zymography detecting MMP-2/9 activity (C) , double-stained images of co-localization (yellow) of CD11C (green) and CD68 (red) -positive areas (D) and double-stained images of co-localization (yellow) of CD206 (green), and CD68 (red) -positive areas (D) . Scale bars, 50 μm. Bar charts show quantification of collagen, α-actin area in the atherosclerotic lesion, MMP-2/9 activity, weighted colocalization coefficients for CD11C and CD206 positive areas in baseline, control, and HCP1-treated groups. Data are mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; n = 6. Statistical analyses were performed using one-way ANOVA.

Article Snippet: Antibodies against c-Myc (sc-40), CD31 (sc-1506), α-actin (sc-32251), CD11C (sc-28671), CD206 (sc-58987), CD68 (sc-7084), normal mouse IgG (sc-2025), and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Staining, Immunostaining, In Situ, Zymography, Activity Assay, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Iba1 + (microglia/monocyte lineage), CD68 + (macrophages) and MPO + (neutrophils) cells presence in the olfactory epithelium before and during SARS-CoV-2 infection. Immunostaining on successive slides of the olfactory epithelium from a non-infected ( A ) or 1 dpi hamster ( B ). Only Iba1 + cells are present in the uninfected olfactory epithelium (OE) and in the lamina propria (LP). In the infected epithelium, Iba1 + cells are massively present in the OE while CD68 + and MPO cells are mostly present in the desquamated cells (red asterisk) in the lumen of the nasal cavity (white asterisk)

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Immunostaining

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: CD68 + macrophage and MPO + neutrophil cells are associated with damage of the olfactory epithelium during SARS-CoV-2 infection. CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (OE, left) and lamina propria (LP, right) in either control animals (CTL) or at 1 or 2 days post-infection (dpi) (Mean normalized to control ± SEM, n = 4, * p < 0.05 (Mann–Whitney test)). Correlation between score damage and percentage of CD68 + ( A 1 ) and MPO + ( B 2 ) signal in the olfactory epithelium (left panel) and the lamina propria (right panel). Spearman test p value

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Control, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Neutrophils play a major role in the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

doi: 10.1007/s00018-022-04643-1

Figure Lengend Snippet: Immunosuppression induced by cyclophosphamide reduces damage of the olfactory epithelium as well as OE infection area. ( A ) Expression of innate immune genes in the nasal turbinates with or without cyclophosphamide treatment at 1 and 2 days post-infection (dpi). Iba1, CD68 and Ncf2 are related to the presence of microglia/macrophages, monocytes/macrophages and neutrophils, respectively; TNFα and IL6 are two cytokines expressed during inflammation; SARS-CoV-2 N expression is related to the SARS-CoV-2 infection. Results represent the Mean ± SEM relative to vehicle-treated hamsters ( n = 4, * p < 0.05; Mann–Whitney test). Representative images of the infected olfactory epithelium immunostained for MPO (neutrophil marker) and SARS-CoV-2 N protein in ( B ) vehicle and ( C ) cyclophosphamide treated animal (olfactory epithelium (OE), lamina propria (LP)). In the vehicle condition, the lumen (white asterisk) is filled with desquamated cells (red asterisk) containing MPO signal. In the cyclophosphamide condition, MPO signal is absent and the lumen is mostly free of cellular debris . Quantification in the OE of ( D 1 ) MPO + neutrophil presence ( D 2 ) damage score ( D 3 ) SARS-CoV-2-infected area and in the lumen of the nasal cavity of ( D 4 ) desquamated cells area and ( D 5 ) percentage of SARS-CoV-2-infected area in the desquamated cells (Mean ± SEM, n = 8 areas of the nasal cavity from 4 different animals, * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann–Whitney))

Article Snippet: The sections were then incubated overnight with primary antibodies directed against SARS nucleocapsid protein (1/500; mouse monoclonal; clone 1C7C7; Sigma-Aldrich), ionized calcium-binding adapter molecule 1 (Iba1) (1/500; rabbit monoclonal; clone EPR16588; Abcam), myeloperoxidase protein (MPO) (1/500; rabbit monoclonal; clone EPR20257; Abcam), CD68 (1/200; rabbit polyclonal; PA1518; Boster), cleaved caspase 3 (C3C) (1/200; rabbit polyclonal; #9661; Cell signaling), G olf (1/300; rabbit polyclonal; C-18; Santa Cruz), Olfactory Marker Protein (OMP) (1/500; goat polyclonal; 544-10001; Wako) and CK18 (1:50; mouse polyclonal; MAB3234—RGE53, Sigma-Aldrich).

Techniques: Infection, Expressing, MANN-WHITNEY, Marker