Journal: bioRxiv

Article Title: Tumor-associated macrophages enhance tumor innervation and spinal cord repair

doi: 10.1101/2024.12.19.629374

Figure Lengend Snippet: A) Gene set enrichment analyses of “neural growth” gene list in mouse M2 and TAM in vitro -generated (n=3/condition), showing enriched expression in TAM groups. B) Immunofluorescence images of human iPSC-derived MNs (CTRL) alone or cocultured with TAM tdTomato or M2 tdTomato (red), stained with the neurite marker β3Tub (green) and DAPI for nuclei (blue). Scale bar=50µm. C) Barplot showing increased β3Tub expression in iPSC-derived MNs treated with TAM (CTRL and M2: n=4/condition; TAM: n=3). D) Barplot showing increased β3Tub expression in mNSCs treated with TAM compared to all other conditions (CTRL: n=4; TAM: n=5; M2: n=3). E, F) Immunofluorescence images of (E) mNSCs (CTRL) or (F) DRG, cocultured with either TAM, or M2 co-cultures, stained with β3Tub (green) and DAPI (blue). CD68 + TAM and M2 macrophages are shown in red. Scale bar=50µm. G) Barplots showing increased β3Tub expression DRGs treated with TAM (CTRL: n=4; TAM: n=3). H-J) Barplots showing the quantification of the lesion extension in 2 days post fertilization (dpf) injured Tg(-3.1 neurog1 :GFP) sb2 and injured TAM-treated Tg(-3.1 neurog1 :GFP) sb2 zebrafish embryos at 1 day post injury (dpi) (H), and 2dpi (I), highlighting a smaller lesion in TAM-treated embryo (Injured: n=14; Injured +TAM: n=23 at 1dpi; Injured: n=10; Injured: +TAM: n=17 at 2 dpi). Representative immunofluorescence images of Tg(-3.1 neurog1 :GFP) sb2 zebrafish embryos in healthy, injured, and injured TAM-treated condition in J. GFP-neurogenin is shown in green, and TAM are shown in red. Scale bar=100µm. K) Confocal immunofluorescence maximum intensity projections of z-stack images of mNSCs (CTRL), cocultured with either TAM, or with TAM LV Scramble , and TAM LV Spp1-KD stained with β3Tub (white), and DAPI (blue). Transduced TAM are shown in green. Scale bar=50µm. L) Barplot showing the increased β3Tub expression (fold change respect to CTRL) in mNSCs treated with TAM LV Scramble , which was inhibited when TAM LV Spp1-KD were used (CTRL and +TAM LV Scramble : n=6/condition; +TAM: n=8; +TAM LV Spp1-KD : n=5). M) Barplot showing the increased β3Tub expression (fold change respect to CTRL) in mNSCs treated with TAM, which was inhibited with Parecoxib, but not Celecoxib, administration (CTRL: n=6; +TAM and +TAM+Parecoxib: n=8/condition; +TAM+Celecoxib: n=3). N) Confocal immunofluorescence maximum intensity projections of z-stack images of mNSCs (CTRL), cocultured with TAM with or without either Parecoxib or Celecoxib, stained with β3Tub (white) and DAPI (blue). Scale bar=50μm. O) Representative western blot and quantification (P) showing the increase β3Tub expression in mNSCs (CTRL) when cocultured with TAM, and the inhibition of this effect following Parecoxib administration. Proteins are indicated by their specific molecular weights (β3Tub, 55kDa) (n=5/condition). Q) Confocal immunofluorescence maximum intensity projections of z-stack images of iPSC-derived MNs (CTRL), iPSC-derived MNs +JRAB2011, iPSC-derived MNs +TAM, and iPSC-derived MNs +TAM+JRAB2011 co-cultures, stained with β3Tub (green), CD68 (red), and DAPI (blue). Scale bar=50µm. R) Barplot showing the increase of β3Tub expression in iPSC-derived MNs cocultured with TAM, which was inhibited following JRAB2011 administration (n=4/condition). S) Scramble and mNSC LV Rictor-KD co-cultured w/o TAM stained with β3Tub (green), CD68 (red), and DAPI (blue). Scale bar=50µm. T) Barplot showing the increase of β3Tub expression in mNSC LV Scramble cocultured with TAM, which was inhibited with mNSC LV Rictor-KD (n=4/condition). U) Simplified schematic representation of the experimental design of the in vitro experiments in . TAM induced neural growth and differentiation in NSC. The pharmacological (Parecoxib)/lentiviral inhibition of SPP1 in TAM (TAM LV SPP1-KD ) inhibit TAM-induced neural growth and differentiation. The pharmacological (JRAB2011)/lentiviral inhibition of Rictor in NSC (NSC LV Rictor-KD ) inhibit TAM-induced neural growth effetc. Data are expressed as mean±SEM. Statistical differences were evaluated by unpaired Student t-test or 1-way ANOVA.

Article Snippet: hTAM (2 x 10 5 cells) were stained with the following antibodies: Anti-human CD14, VioBlue REAfinity (Miltenyi Biotec); Anti-human CD68, APC REAfinity (Miltenyi Biotec); Anti-human CD163, APC-Vio770 REAfinity (Miltenyi Biotec); Anti-human CXCR4, VioBright FITC REAfinity (Miltenyi Biotec); Anti-human HIF-1A, Alexa Fluor 647 (BD Biosciences); Anti-human VEGFA, PE (Abcam); Anti-hOsteopontin, APC (R&D).

Techniques: In Vitro, Generated, Expressing, Immunofluorescence, Derivative Assay, Staining, Marker, Western Blot, Inhibition, Cell Culture

Journal: Nature Communications

Article Title: Modeling alpha-synuclein pathology in a human brain-chip to assess blood-brain barrier disruption

doi: 10.1038/s41467-021-26066-5

Figure Lengend Snippet: a Experimental design for assessing the effects of αSyn toxic aggregates (fibrils) in the Substantia Nigra Brain-Chip, including the seeding in the Brain-Chip, the timeline for medium changes, as well as sampling times. b Immunofluorescence micrographs show the accumulation of phosphorylated αSyn (green, phospho-αSyn129 staining; blue, DAPI) at day 6 post-exposure (D8). Pathology is absent in the brain channel following exposure to monomer or PBS. Scale bars: 100 μm. c Quantitative analysis of fluorescence intensity in each group at day 3 and 6 post-exposure (D5 and D8, respectively). Statistical analysis is two-way ANOVA with Tukey’s multiple comparisons test ( n = 3–4 independent chips with 3~5 randomly selected different areas per chip, * P = 0.0103, **** P < 0.0001 compared to monomeric group). Error bars represent mean ± SEM. d Western blotting analysis of cell lysates from the brain channel shows significant intracellular phosphorylation of αSyn at Ser129 (MW: 18 kDa) following exposure to αSyn fibrils, whereas there was no effect upon exposure to the PBS. For loading control, equal amounts of protein were immunoblotted with GAPDH antibody (MW: 37 kDa). e Confocal images of double immunostaining for phospho-αSyn129 (green) and tyrosine hydroxylase (red, TH), in the brain channel at day 6 post-exposure (D8) to αSyn fibrils (white arrow). Scale bars: 50 μm. f , g αSyn fibrils are taken up by astrocytes and microglia (white arrow), as evidenced by double immunostaining for phospho-αSyn129 (green) and either glial fibrillary acidic protein (red, GFAP) for astrocytes or ionized calcium-binding adaptor molecule 1 (red, IBA1) for microglia in the brain channel at day 6 post-exposure (D8) to αSyn fibrils. Scale bars: 50 μm.

Article Snippet: The target genes were assessed using commercially available primers ( GFAP : Hs00909233_m1, VIMENTIN : Hs00958111_m1, LCN2 : Hs01008571_m1, CD68 : Hs02836816_g1, EDA : Hs03025596_s1, APOA1 : Hs00163641_m1, SNCAIP : Hs00917422_m1, LRRK2 : Hs01115057_m1, MAOA : Hs00165140_m1).

Techniques: Sampling, Immunofluorescence, Staining, Fluorescence, Western Blot, Phospho-proteomics, Control, Double Immunostaining, Binding Assay

Journal: Nature Communications

Article Title: Modeling alpha-synuclein pathology in a human brain-chip to assess blood-brain barrier disruption

doi: 10.1038/s41467-021-26066-5

Figure Lengend Snippet: a Representative merged images showing immunostaining for microtubule-associated protein 2 (gray, MAP2), tyrosine hydroxylase (red, TH), and Cleaved Caspase-3 (green, CC3) in the brain channel at 6-days post-exposure to αSyn fibrils or αSyn monomers. Scale bars: 100 μm. b Quantitation of the number of CC3 and MAP2- or TH-positive neurons. Statistical analysis by two-way ANOVA with Tukey’s multiple comparisons test (3–4 randomly selected different areas per chip, n = 3 independent chips/experimental group, ** P = 0.0033, **** P < 0.0001, NS not significant). Error bars represent mean ± SEM. Scale bars: 100 μm. c Immunostaining of the astrocyte marker glial fibrillary acidic protein (magenta, GFAP) demonstrated activation of astrocytes (white arrow) at day 6 post exposure to αSyn fibrils compared to monomeric αSyn. Scale bar, 100 μm. d Immunostaining of the microglial CD68 (red) demonstrated activation of microglia (white arrow) at day 6 post exposure to αSyn fibrils compared to monomeric αSyn. Scale bar, 100 μm. e The secreted levels of tumor necrosis factor-alpha (TNF-α) in the αSyn fibril model. Statistical analysis is two-sided unpaired t -test ( n = 6–7 independent chips, ** P = 0.0023). Error bars represent mean ± SEM. f The secreted levels of proinflammatory cytokine interleukin-6 (IL-6) in the αSyn fibril model. Statistical analysis is two-sided unpaired t -test ( n = 4–7 independent chips, **** P < 0.0001). Error bars represent mean ± SEM.

Article Snippet: The target genes were assessed using commercially available primers ( GFAP : Hs00909233_m1, VIMENTIN : Hs00958111_m1, LCN2 : Hs01008571_m1, CD68 : Hs02836816_g1, EDA : Hs03025596_s1, APOA1 : Hs00163641_m1, SNCAIP : Hs00917422_m1, LRRK2 : Hs01115057_m1, MAOA : Hs00165140_m1).

Techniques: Immunostaining, Quantitation Assay, Marker, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake *

doi: 10.1074/jbc.M115.646240

Figure Lengend Snippet: PCPE2 deficiency exacerbates atherosclerosis despite elevated HDL cholesterol levels. A, representative aortic root sections stained with Oil Red O from 12 weeks atherogenic diet-fed LDLr−/−, LDLr−/− PCPE2−/−, and LDLr−/−, apoA-I−/− mice. B and C, quantification of the atherosclerotic lesion area as a percentage of the total aortic area (B) and the total lesion area in μm2 (C). D, representative aortic root sections stained with fluorescently labeled antibodies to CD68, a macrophage marker, from 12 weeks atherogenic diet-fed LDLr−/− mice, LDLr−/−,PCPE2−/− mice, and LDLr−/−, apoA-I−/− mice. E and F, quantification of the macrophage content was performed by measuring CD68+ staining over background as a percentage of the total lesion area (E) and of the total lesion area in μm2 (F). Data represent the mean ± S.D. of n = 10 male mice/group. Different letters indicate statistical significance at p < 0.05. The fluorescence background threshold was set to the intensity of sections receiving the fluorescently tagged secondary antibody but no CD68 primary antibody.

Article Snippet: For immunofluorescence microscopy, purified rat anti-mouse CD68 (AbD FA-11-Serotec) at a 1:100 dilution was used as the primary antibody.

Techniques: Staining, Labeling, Marker, Fluorescence

Journal: Journal of Inflammation Research

Article Title: Histone Deacetylase 6 Regulates the Activation of M1 Macrophages by the Glycolytic Pathway During Acute Liver Failure

doi: 10.2147/JIR.S302391

Figure Lengend Snippet: The effect of ACY-1215 on cytokines and marker proteins of M1 macrophages in ALF mice. ( A – D ) The levels of HIF1α, TNF-α, IL-6, and IL-1β were tested by ELISA kits. ( E ) The levels of iNOS and CD68 were detected by immunofluorescence. Data are shown as mean ± SD. # P < 0.05, compared with the control group. * P < 0.05, compared with the LPS/D-Gal group.

Article Snippet: Rabbit anti-mouse inducible nitric oxide synthase (iNOS, Cat. No. 18985-1-AP) and CD68 (Cat. No. 28058-1-AP) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Marker, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Control

Journal: International Journal of Molecular Medicine

Article Title: Effects of ketanserin on experimental colitis in mice and macrophage function

doi: 10.3892/ijmm.2016.2486

Figure Lengend Snippet: 5-Hydroxytryptamine-2A receptor (5-HT 2A R) expression is upregulated in the colons of patients with inflammatory bowel disease (IBD) and in mice with dextran sodium sulfate (DSS)-induced colitis, and is specifically enhanced in macrophages. (A) Relative mRNA expression of 5-HT 2A R in healthy subjects and patients wtih IBD estimated by RT-qPCR. (B) Representative images of immunofluorescence staining of colonic mucosa samples of healthy subjects and in patients with IBD showing 5-HT 2A R + cells (in green) and CD68 + cells (in red). Co-localization of 5-HT 2A R with the macrophage marker, CD68 + , is shown in the merged images. 5-HT 2A R expression in mice with DSS-induced colitis estimated by (C) RT-qPCR and (D) western blot analysis. Representative images of immunofluorescence staining of 5-HT 2A R + cells (in green) and CD68 + cells (in red) in the colonic mucosa samples of mice treated with or without DSS. (E) Co-localization of 5-HT 2A R with CD68 is shown in the merged images.

Article Snippet: Mouse anti-human CD68 antibody (MCA5709; mouse anti-human CD68, monoclonal antibody; AbD Serotec, Kidlington, UK; 1:500) (overnight at 4°C) was subsequently used to detect the macrophages.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Marker, Western Blot

Journal: Life

Article Title: Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling

doi: 10.3390/life14081031

Figure Lengend Snippet: Time course of the C5a/C5aR1 axis, expression in rat kidney sections, C5a concentration in rat plasma and numbers of renal macrophages. The left kidneys were harvested after I/R at the following time points: 10 min, 6 h, 24 h, 3 d, 5 d and 8 w. The healthy right kidney was nephrectomized one week before and served as a control. mRNA was isolated from fresh-frozen rat kidney tissue. C5a was measured in rat plasma using an ELISA ( A ). Plasma C5a concentrations were correlated with serum urea ( B ). Expression of C5ar1 ( C ) and C5 ( D ). mRNA was analyzed at different time points post-perfusion. C5ar1 mRNA expression correlated with the expression of the macrophage marker Cd68 ( E ), while no correlation was found between Cd68 expression and plasma C5a levels ( F ). Immunohistochemistry was used for the evaluation of CD163-positive ( G ) and CD68-positive macrophages in kidney sections ( H ) (ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The following primary antibodies were used: mouse anti-human α-SMA, mouse anti-human Vimentin (both from DAKO Deutschland, Hamburg, Germany, and cross-reactive against rats), mouse anti-rat CD163 and mouse anti-rat CD68 (clone ED1) (both from Bio-Rad Laboratories GmbH, Feldkirchen, Germany).

Techniques: Expressing, Concentration Assay, Clinical Proteomics, Control, Isolation, Enzyme-linked Immunosorbent Assay, Marker, Immunohistochemistry

Journal: Life

Article Title: Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling

doi: 10.3390/life14081031

Figure Lengend Snippet: C5ar1 and C3ar1 in situ hybridization and immunofluorescence co-localization studies in I/R rat kidney sections. 4 µm sections of FFPE kidneys from d3 post-reperfusion were utilized and processed according to the RNAscope standard protocol. C5ar1 and C3ar1 probes were conjugated with fluorochromes, and after assay completion, the sections were incubated with fluorescent antibodies against UMOD ( A , C , D ) and PDGFRB ( A , D ) or against CD68 ( B ).

Article Snippet: The following primary antibodies were used: mouse anti-human α-SMA, mouse anti-human Vimentin (both from DAKO Deutschland, Hamburg, Germany, and cross-reactive against rats), mouse anti-rat CD163 and mouse anti-rat CD68 (clone ED1) (both from Bio-Rad Laboratories GmbH, Feldkirchen, Germany).

Techniques: In Situ Hybridization, Immunofluorescence, RNAscope, Incubation

Journal: Molecular cancer research : MCR

Article Title: PRMT6 promotes lung tumor progression via the alternate activation of tumor-associated macrophages

doi: 10.1158/1541-7786.MCR-19-0204

Figure Lengend Snippet: A. Lung tumor sections of PRMT6Tg; Sftpc-Cretm and PRMT6Tg control mice after tamoxifen and urethane treatment (20 weeks) were subjected to indirect immunofluorescence staining with anti-CD68, anti-iNOS, and anti-arginase I antibodies as described in the methods. Scale bar: 20 μM. B. Gating strategy for the purification of TAMs using CD11b antibodies. Sixty% of live cells are CD11b+, of which 83% are F4/80+. C. Total RNA from CD11b+ cells from the lungs of PRMT6Tg; Sftpc-Cretm and PRMT6Tg control mice after tamoxifen and urethane treatment (20 weeks) were employed in qPCR analysis for the indicated M1 and M2 macrophage markers. *, p< 0.05; versus control. D. Bone marrow-derived macrophages were treated with the conditioned medium isolated from AECs isolated from PRMT6Tg; Sftpc-Cretm or PRMT6Tg control mice after tamoxifen treatment. Later, total RNA from the macrophages were isolated and qPCRs were performed for M1 and M2 macrophage markers as described in the methods. **, p< 0.01; *, p< 0.05; versus control. E. THP1 cells were incubated with 150 mM phorbol myristate acetate (PMA) for 24h. Later, the differentiated macrophages were incubated with conditioned medium from H2122 PRMT6 knockout clone or parental cells for additional 24 h. Total RNA from the macrophages were isolated and qPCRs were performed for M2 macrophage marker as described in the methods. **, p< 0.01. F. Total RNA isolated from bone marrow-derived macrophages treated with the conditioned medium isolated from AECs isolated from PRMT6Tg; Sftpc-Cretm or PRMT6Tg control mice after tamoxifen treatment were employed in qPCRs for pro-angiogenic gene markers as described in the methods. *, p< 0.05; versus control. G. Total RNA isolated from lungs of PRMT6Tg; Sftpc-Cretm and PRMT6Tg control mice after tamoxifen and urethane treatment (20 weeks) were subjected to qPCR analysis of pro-angiogenic gene markers as described in the methods. **, p< 0.01 *, p< 0.05; versus control. H. Lung tumor sections of PRMT6Tg; Sftpc-Cretm and PRMT6Tg control mice after tamoxifen and urethane treatment (20 weeks) were subjected to indirect immunofluorescence staining with anti-CD31 antibodies as described in the methods. Scale bar: 10 μM.

Article Snippet: Indirect immunofluorescence Lung tumor sections from PRMT6 Tg ; Sftpc-Cre tm and PRMT6 Tg mice 20 weeks after tamoxifen and urethane treatment were employed in immunostaining procedure using the following antibodies: CD68 (FA-11, Bio-Rad) for identifying macrophages, iNOS (ab15323, Abcam) to detect M1 polarization, Arginase I (sc-18351, Santa Cruz Biotechnology) to detect M2 polarization, EPCAM (VU1D9, Cell signaling technology), alpha smooth muscle actin (ab5694, Abcam), and CD31 (550274, BD Biosciences) using procedures described previously ( 49 ).

Techniques: Immunofluorescence, Staining, Purification, Derivative Assay, Isolation, Incubation, Knock-Out, Marker

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Increased Atherosclerotic Lesions in LDL Receptor Deficient Mice With Hematopoietic Nuclear Receptor Rev‐erbα Knock‐ Down

doi: 10.1161/JAHA.113.000235

Figure Lengend Snippet: Rev‐erbα protein is present in the macrophages of human carotid artery atherosclerotic lesions. Human carotid artery sections were subjected to immunohistochemistry using mouse monoclonal anti‐human Rev‐erbα. A , Rev‐erbα immunoreactivity in a representative plaque (40×) (left panels), and a high‐magnification view of the same image (400×) (right panels) illustrating the cellular nature of the staining. B , Immunofluoresence staining identifying the Rev‐erbα positive cells as macrophages. A representative section of a human carotid plaque stained by using primary antibodies against Rev‐erbα (green) and the macrophage‐specific marker CD68 (red). Colocalization of the two markers is shown in yellow (left panels), and a higher magnification view of the same immunofluorescence image (right panels).

Article Snippet: After copious washing in PBS, the sections were incubated with fluorescently labeled secondary antibodies diluted in 1% (w/v) BSA in PBS for 30 minutes at 37°C (Cy3 conjugated goat antirabbit IgG [Proteintech Group Inc] for detection of CD68 antibodies; Alexa Fluor 488 conjugated goat anti‐mouse IgG [Invitrogen] for detection of Rev‐erbα antibodies).

Techniques: Immunohistochemistry, Staining, Marker, Immunofluorescence

Journal: Journal of vascular research

Article Title: Myeloid lineage of human endothelial outgrowth cells circulating in blood and vasculogenic endothelial-like cells in the diseased vessel wall.

doi: 10.1159/000226226

Figure Lengend Snippet: Fig. 1. Characterisation of EOC. FACS analysis of haematopoietic, angioblastic and myeloid markers. EOC ( a ) expression of monocytic markers CD68, Mac-1 and to a lesser extent CD14, but lack of the pan- leukocyte marker CD45. EOC were posi- tive for Flk1, eNOS, VECAD and Tie-2 ( a ). In contrast, MNC were negative for endo- thelial markers eNOS, VECAD and Tie-2 ( b ). c Analysis of endothelial marker tran- script expression in EOC, SOC and HAEC by RT-PCR.

Article Snippet: The primary antibodies used were mouse monoclonal antibodies against Mac-1 (CD11b, Ancell, 159-020), CD31 (Dako, M0823), CD34-PerCP (for FACS, BD Pharmingen, BDEurope, 345804), CD34 (for immunofluorescence, Santa Cruz, sc-7324), Flk-1 (Santa Cruz, sc-6251), CD45 (Santa Cruz, sc-1178), rabbit polyclonal antibodies against CD68 (Santa Cruz, sc-9139), Tie-2 (Santa Cruz, sc-324) and goat polyclonal antibodies against CD14 (Santa Cruz, sc-6997).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of vascular research

Article Title: Myeloid lineage of human endothelial outgrowth cells circulating in blood and vasculogenic endothelial-like cells in the diseased vessel wall.

doi: 10.1159/000226226

Figure Lengend Snippet: Fig. 2. a Primary outgrowth colony forma- tion following single-cell deposition, ex- panding colony and confluent monolayer by phase contrast microscopy. b FACS analysis showed positivity with endothe- lial markers (eNOS, VECAD, Tie-2 and Flk-1) and some myeloid markers (CD68 and Mac-1). G3PDH shows equivalent RNA loading conditions.

Article Snippet: The primary antibodies used were mouse monoclonal antibodies against Mac-1 (CD11b, Ancell, 159-020), CD31 (Dako, M0823), CD34-PerCP (for FACS, BD Pharmingen, BDEurope, 345804), CD34 (for immunofluorescence, Santa Cruz, sc-7324), Flk-1 (Santa Cruz, sc-6251), CD45 (Santa Cruz, sc-1178), rabbit polyclonal antibodies against CD68 (Santa Cruz, sc-9139), Tie-2 (Santa Cruz, sc-324) and goat polyclonal antibodies against CD14 (Santa Cruz, sc-6997).

Techniques: Microscopy

Journal: Journal of vascular research

Article Title: Myeloid lineage of human endothelial outgrowth cells circulating in blood and vasculogenic endothelial-like cells in the diseased vessel wall.

doi: 10.1159/000226226

Figure Lengend Snippet: Fig. 4. Immunostaining of CD68 and costaining of CD68 with endothelial-specific antigens in microvessels within the adventi- tia of diseased coronary arteries (please note: colours are shown in the online version only). a Immunostaining of CD68+ cells (ar- row; red) lining adventitial microvessel. Note erythrocytes (white; open arrowhead) within the microvessel lumen (L). b Multiple CD68+ cells (red) in microvessels within the adventitia of a me- dium-sized diseased artery. c Recipient-derived Y-chromosome- positive (green dot in blue nucleus) CD68+ (red) cell in adventitial microvessel (arrow) of a vasculopathic coronary artery from a pa-

Article Snippet: The primary antibodies used were mouse monoclonal antibodies against Mac-1 (CD11b, Ancell, 159-020), CD31 (Dako, M0823), CD34-PerCP (for FACS, BD Pharmingen, BDEurope, 345804), CD34 (for immunofluorescence, Santa Cruz, sc-7324), Flk-1 (Santa Cruz, sc-6251), CD45 (Santa Cruz, sc-1178), rabbit polyclonal antibodies against CD68 (Santa Cruz, sc-9139), Tie-2 (Santa Cruz, sc-324) and goat polyclonal antibodies against CD14 (Santa Cruz, sc-6997).

Techniques: Immunostaining, Derivative Assay

Journal: Journal of vascular research

Article Title: Myeloid lineage of human endothelial outgrowth cells circulating in blood and vasculogenic endothelial-like cells in the diseased vessel wall.

doi: 10.1159/000226226

Figure Lengend Snippet: Fig. 5. Double immunostaining of CD68 and Tie-2 in cells lining microvascular channels within the adventitia and the lu- men (L) of medium-sized diseased arteries (please note: colours are shown in the on- line version only). a Longitudinal axis of a microvascular channel (asterisks) in dis- eased vessel adventitia showing costaining of CD68 (green) and Tie-2 (red). The merged image shows combined CD68 and Tie-2 staining (yellow). b Transverse sec- tion through multiple microvascular channels in diseased artery adventitia showing similar CD68/Tie-2 costaining. c Erythrocyte (white; open arrowhead)- filled channel in longitudinal orientation within the adventitia of atherosclerotic vessel showing lining cells co-immunola- belling for CD68 and Tie-2. d , e Mouse (MIgG) and rabbit IgG (RIgG) control an- tibody labelling showing lack of immu- noreactivity. f Double immunolabelling (merged-yellow) of CD68 (green) and Tie- 2 (red) in cells lining the endoluminal sur- face of atherosclerotic vessel. In each pan- el, arrows indicate cells that label positive for CD68 and Tie-2 singly and combined.

Article Snippet: The primary antibodies used were mouse monoclonal antibodies against Mac-1 (CD11b, Ancell, 159-020), CD31 (Dako, M0823), CD34-PerCP (for FACS, BD Pharmingen, BDEurope, 345804), CD34 (for immunofluorescence, Santa Cruz, sc-7324), Flk-1 (Santa Cruz, sc-6251), CD45 (Santa Cruz, sc-1178), rabbit polyclonal antibodies against CD68 (Santa Cruz, sc-9139), Tie-2 (Santa Cruz, sc-324) and goat polyclonal antibodies against CD14 (Santa Cruz, sc-6997).

Techniques: Double Immunostaining, Staining, Control

Journal: Journal of vascular research

Article Title: Myeloid lineage of human endothelial outgrowth cells circulating in blood and vasculogenic endothelial-like cells in the diseased vessel wall.

doi: 10.1159/000226226

Figure Lengend Snippet: Fig. 6. FISH for Y chromosome indicated that CD68/Tie-2 double-positive cells were of recipient in origin. [Online version: red = CD68 and X chromosome (arrow- head); green = Tie-2 and Y chromosome (open arrowhead); blue = DAPI-stained nuclei; yellow = merged red (CD68) and green (Tie-2)]. The recipient-derived male cells are diploid as indicated by two sex chromosomes (arrow) and show no evi- dence of cell fusion. L = Lumen.

Article Snippet: The primary antibodies used were mouse monoclonal antibodies against Mac-1 (CD11b, Ancell, 159-020), CD31 (Dako, M0823), CD34-PerCP (for FACS, BD Pharmingen, BDEurope, 345804), CD34 (for immunofluorescence, Santa Cruz, sc-7324), Flk-1 (Santa Cruz, sc-6251), CD45 (Santa Cruz, sc-1178), rabbit polyclonal antibodies against CD68 (Santa Cruz, sc-9139), Tie-2 (Santa Cruz, sc-324) and goat polyclonal antibodies against CD14 (Santa Cruz, sc-6997).

Techniques: Staining, Derivative Assay

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a Overview of the workflow and discovery/validation cohorts. OA osteoarthritis, RA rheumatoid arthritis, s single sample, p paired samples. Created in BioRender. Xu, H. (2025) https://BioRender.com/o60h131 . b Visualization of nine main clusters across 63,035 cells using t-distributed stochastic neighbor embedding (tSNE). BT before treatment, AT after treatment. c Dot plot illustrating the expression level of marker genes across SF clusters. The dots’ size and color spectrum indicate positive percentage and average expression (log1p transformed) of particular markers genes in each cell type, respectively. d Quantification of absolute cell count and relative proportions of distinct cell clusters in SF among patients. The horizontal coordinates depict cell number and proportions, the vertical coordinates depict patients. e Density of macrophage clusters among different groups. Density is calculated by multiplying the total cell density in each SF sample by the proportion of macrophage cluster in the sample. The p -values were calculated using a two-sided Wilcoxon test. OA-BT: n = 3, RA-BT: n = 6, and RA-AT: n = 6. solid squares, patients didn’t receive b/tsDMARDs treatment; hollow circles, adalimumab treated; solid circles, tofacitinib treated. Data are presented as mean values ± SEM. f Deconvolution of bulk RNA-seq data for eight major cell clusters across all samples based on canonical marker genes. The p -values were calculated using a two-sided Wilcoxon test, comparing OA-BT ( n = 5), RA-BT ( n = 14), and RA-AT ( n = 10). The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value. g Left: Representative flow cytometry (FACS) dot plots of CD64 and CD11b expression in SF cells. Right: Comparison of FACS-based macrophage proportions. Data are presented as mean ± SD. p -values were calculated using a two-sided Student’s t test. h Deconvolution of published bulk data (GSE55235) for macrophages in ST samples from OA ( n = 10) and RA ( n = 10) patients. The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value.

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Biomarker Discovery, Expressing, Marker, Transformation Assay, Cell Counting, RNA Sequencing, Flow Cytometry, Comparison

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a Heatmap depicting the expression positivity of well-reported RA-related genes in the major cell types. The color spectrum signifies the ratio of cells that exhibit positive gene expression. b Dot plot illustrating the pairwise comparisons of expression levels of well-reported RA-related genes in different cell clusters. The p -values were calculated using a two-sided Wilcoxon test. The dot size and color spectrum indicate q -value (-log10 transformed) and fold change (log2 transformed) of gene expression, respectively. c Feature plot showing the count of differentially expressed genes (DEGs) determined by a two-sided Wilcoxon test. d Volcano plot displaying the DEGs in macrophages. The adjusted p -values were calculated using a two-sided Wilcoxon test. N.S., non-significant ( p > 1 × 10 −10 ); Sig, significant ( p < 1 × 10 −10 ); Overlapped-up and -down indicate the intersecting DEGs significantly up- and down-regulated in RA-BT group ( p < 1 × 10 −10 ), respectively. e , f Enriched gene ontology (GO) and gene set enrichment analysis (GSEA) pathways for the overlapped-up genes in macrophages from the RA-BT group. p -values were calculated by the one-sided Permutation test. g Volcano plot displaying the DEGs in T cells, similar to d . h Enriched GO pathways for the overlapped-up genes in T cells from RA-BT. i , j DEGs in macrophage and T cell clusters between the ACR20_N ( n = 2882 macrophages; n = 985 T cells) and ACR20_Y ( n = 12,658 macrophages; n = 5904 T cells) groups. The p -values were calculated by the two-sided Wilcoxon test, **** p < 2.2 × 10 −16 . The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value.

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Expressing, Gene Expression, Transformation Assay

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a tSNE plot of macrophage subclusters. b Heatmap of the marker gene expression for each macrophage subtype. c Histogram depicting the distribution of each patient with OA and RA among different subclusters of macrophages. d Density of SPP1 + /S100A12 + macrophages among patient groups. Density is calculated by multiplying the total cell density in each SF sample by the proportion of SPP1 + /S100A12 + macrophage cluster in the sample. The p -values were computed using the two-sided Wilcoxon test for comparisons between OA-BT ( n = 3), RA-BT ( n = 6), and RA-AT ( n = 6). solid squares, patients didn’t receive b/tsDMARDs treatment; hollow circles, adalimumab treated; solid circles, tofacitinib treated. Data are presented as mean values ± SEM. e Correlation of SPP1 + macrophages proportion and DAS28. hollow circles, adalimumab treated; solid circles, tofacitinib treated. p -values were calculated using two-sided Pearson correlation test. f The different expression levels of SPP1 and CCL2 in SPP1 + macrophage among different groups. p -values were calculated by the two-sided Wilcoxon test for comparisons between OA-BT ( n = 1715 cells), RA-BT ( n = 10,207 cells), and RA-AT ( n = 5059 cells), **** p < 2.2 × 10 −16 . The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value. g Different level of secreted osteopontin (encoded by SPP1 ) and CCL2 evaluated by enzyme-linked immunosorbent assay (ELISA) in SF. solid squares, patients didn’t receive b/tsDMARDs treatment; hollow circles, adalimumab treated; solid circles, tofacitinib treated. OA-BT: n = 10; RA-BT: n = 27; RA-AT: n = 12. The p -values were calculated by the two-sided Wilcoxon test. Data are presented as mean values ± SEM. h Pearson’s correlation analysis of secreted osteopontin levels and DAS28 in validation cohort. p -values were calculated using two-sided Pearson correlation test. i Comparison of the levels of osteopontin and CCL2 in ACR20_Y ( n = 12) vs . ACR20_N ( n = 8). The p -values were calculated by the two-sided Wilcoxon test. Data are presented as mean values ± SEM.

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Marker, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Comparison

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a Upper left: schematic representation of coculture experiments involving sorted macrophages and fibroblast-like synoviocytes (FLS); Lower left: representative FACS plot of CD11b + CD64 + CD36 + CD62L - cell (representing SPP1 + macrophage), CD11b + CD64 + CD62L + cell (representing S100A12 + macrophage), and CD11b + CD64 + CD36 - CD62L - cell (representing double negative macrophage) from SF of RA patients ( n = 10); Right: mRNA expression of IL-6 , MMP-1, MMP−2 and MMP-13 in FLS, FLS + SPP1 + macrophage, FLS + S100A12 + macrophage, FLS + double negative macrophage through direct coculture system and Trans-well-based coculture system; Data are presented as mean ± SEM. The p -values were calculated by the two-sided Wilcoxon test. FLS fibroblast-like synoviocytes. Created in BioRender. Xu, H. (2025) https://BioRender.com/m45b690 . b Schedule of injection with type II collagen (CII), Freund’s complete adjuvant (CFA), Freund’s incomplete adjuvant (IFA) and sample collection. Created in BioRender. Xu, H. (2025) https://BioRender.com/r59d731 . c Representative images of wrist joint. d Clinical score of collagen-induced arthritis (CIA) between Spp1 -cKO and Spp1 -WT mice. n = 5 per group. p -values were calculated by the two-sided Student’s t test, * p < 0.05, ** p < 0.01. Exact p -values for days 29, 31, 33, and 35 were 0.037, 0.0085, 0.049, and 0.048, respectively. e mRNA expression of II6, Mmp13, Mmp1a in hind paw of Spp1 -cKO ( n = 5) and WT ( n = 5) mice. Data are presented as mean ± SEM. The p -values were calculated by the two-sided Wilcoxon test.

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Expressing, Injection, Adjuvant

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a Heatmap of the monocyte, M1, and M2 enrichment scores. The wide range of hues symbolizes the pathway activities. b Differences in M1 signature scores of SPP1 + / S100A12 + macrophages. p -values were calculated by the two-sided Wilcoxon test, **** p < 2.2 × 10 −16 . c Enrichment of the M1 signature using in-house bulk RNA-seq data for OA-BT ( n = 5), RA-BT ( n = 14), and RA-AT ( n = 10). p- values were calculated by the two-sided Wilcoxon test. The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to the maximum and minimum value. d M1 scores of SPP1 + /S100A12 + macrophages in ACR20_N and ACR20_Y. p -values were calculated by the two-sided Wilcoxon test, **** p < 2.2 × 10 −16 . e Heatmap displaying the single sample gene set enrichment analysis (ssGSEA) results of hallmark gene sets. f Evolutionary trajectory of SPP1 + macrophage in RA patients treated with tofacitinib. Upper, pseudotime curve and activation trajectory; Middle, trajectory and histography of three states; Lower, Different dynamic evolution trajectories between ACR20_Y and ACR20_N. g Dynamic activities of SCENIC-based transcription factors in SPP1 + / S100A12 + macrophages . The dot size and color spectrum indicate q- value (-log10 transformed) and log2 transformed fold change in expression levels of the transcription factors, respectively. p -values were calculated by the two-sided Wilcoxon test. h – i Representative images of multiplex immunofluorescence (mIF) and box plots showing CD68 + SPP1 + STAT1 + and F4/80 + SPP1 + STAT1 + cells in ST samples derived from OA/RA patients and control/CIA mice, respectively. DAPI (blue), SPP1 (red), STAT1 (green), CD68 and F4/80 (yellow), CD68 + SPP1 + STAT1 + and F4/80 + SPP1 + STAT1 + (white arrows) . Scale bars: 20 μm. Box plot illustrating the proportion of CD68 + SPP1 + STAT1 + and F4/80 + SPP1 + STAT1 + cells to total cells in patients with OA ( n = 3) versus RA ( n = 3) and control mice ( n = 3) versus CIA mice ( n = 3), respectively. Data are presented as mean ± SD. The p -values were determined by a two-sided Student’s t test, * p < 0.05, ** p < 0.01. The exact p values CD68 + SPP1 + STAT1 + = 0.018, F4/80 + SPP1 + STAT1 + = 0.036. j Pearson’s correlation of transcription factor expression levels and DAS28/SDAI.

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: RNA Sequencing, Activation Assay, Transformation Assay, Expressing, Multiplex Assay, Immunofluorescence, Derivative Assay, Control

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: a Heatmap of CellphoneDB-based ligand-receptor communication counts among all macrophage and T cell subclusters. b , c Representative images of mIF and box plots showing the interactions between SPP1 + macrophages (patients: CD68 + SPP1 + cells; mice: F4/80 + SPP1 + cells) and CXCL13 + CD4 + T cells (CD3 + CD4 + CXCL13 + cells) in ST samples derived from OA ( n = 3)/RA ( n = 3) patients and control ( n = 3)/CIA ( n = 3) mice, respectively. within 15 μm from the CD3 + CD4 + CXCL13 + T cells were regarded as potential interaction partners. The interaction degree was measured by the proportion of these potential interacting macrophages relative to the total cell population. DAPI (blue), SPP1 (red), CD3 (white), CD4 (cyan), CXCL13 (pink), CD68 and F4/80 (yellow), CD68 + SPP1 + and F4/80 + SPP1 + (red arrows), and CD3 + CD4 + CXCL13 + (green arrows). Scale bars: 10 μm. Data were presented as mean ± SD. The p -values were determined by a two-sided Student’s t test, with ** p < 0.01 and *** p < 0.001. Exact p values OA/RA = 0.00037, Control/CIA = 0.0078. d Heatmap of the fold change in communication counts. e Predicted and detailed CellphoneDB-based ligand-receptor communications. p -value were calculated using a one-sided permutation test in CellPhoneDB. Dot size and color spectrum indicate the p -value (-log10 transformed) and mean expression (log2 transformed) of ligand and receptor, respectively. f iTALK-based Cytokine-chemokine communications among pathogenic macrophage and T cell subclusters. g Left: schematic representation of coculture experiments involving sorted macrophages, CD4 + T cells and fibroblast-like synoviocytes (FLS); Right: mRNA expression of IL-6, MMP-1, MMP-2 and MMP-13 in FLS, FLS + SPP1 + macrophage, FLS + CD4 + T cells, FLS + SPP1 + macrophage+ CD4 + T cells through direct coculture system and Trans-well-based coculture system ( n = 3); Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test for multi-group comparisons. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The exact p values are provided in the Source data. FLS, fibroblast-like synoviocytes. Created in BioRender. Xu, H. (2025) https://BioRender.com/g62b838 .

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Derivative Assay, Control, Transformation Assay, Expressing

Journal: Nature Communications

Article Title: Single cell immunoprofile of synovial fluid in rheumatoid arthritis with TNF/JAK inhibitor treatment

doi: 10.1038/s41467-025-57361-0

Figure Lengend Snippet: The left and right panels represent SF from RA patients before treatment (RA-BT) and after treatment (RA-AT), respectively. In the immunological milieu of SF from patients with RA, activity of various molecular pathways (e.g., JAK/STAT pathway) were increased. Pathogenic macrophages and T cell subtypes produced pro-inflammatory molecules (e.g., CCL2). ligand-receptor interactions were enhanced between pathogenic macrophage subsets (such as SPP1 + /S100A12 + ) and T cell subsets ( CD8 + Tem1, Treg, CXCL13 + CD4 + T cells), along with inter-crosstalk among the three T cell subtypes. These interactions may play a role in the development and advancement of RA and can be blocked in either shared or drug-specific manner. The lighter color of cells in RA-AT indicates lower level of activation of inflammatory genes and RA pathogenic pathways within these cells compared to those in RA-BT. Created in BioRender. Xu, H. (2025) https://BioRender.com/s60q947 .

Article Snippet: For co-colocalization of SPP1 + and CD36 + CD62L - macrophages (CD68 + ), as well as S100A12 + and CD62L + macrophages (CD68 + ) in the ST from RA patient, all antibodies against CD68 (AF20022, Aifang Biological, 1:3000, TYR-520), SPP1(AF03532, Aifang Biological, 1:2000, TYR-570), CD36 (66395-1-Ig, Proteintech, 1:3000, TYR-780), CD62L (222511, ZEN-BIOSCIENCE, 1:2000, TYR-690), and S100A12 (14959, Cell Signaling Technology, 1:2000, TYR-620) were evaluated via immunohistochemistry.

Techniques: Activity Assay, Produced, Activation Assay

Journal: bioRxiv

Article Title: Complement C5a receptor 1 blockade reverses cognitive deficits following cranial radiation therapy for brain cancer

doi: 10.1101/2024.07.02.601806

Figure Lengend Snippet: (A) Dual immunofluorescence staining, confocal microscopy, and 3D algorithm-based, volumetric quantification for the microglial activation (CD68-IBA1 immunoreactivity, magenta-green) in the WT mice show cranial irradiation (9 Gy + Vehicle)-induced elevated CD68 expression by the IBA1 + microglia in the hippocampus ( dh , dentate hilus; dg , dentate gyrus) compared to 0 Gy+Vehicle. (B) A separate CD68-DAPI channel (magenta-blue) is shown for each group. (C) C5aR inhibition (PMX205) in WT mice did not elevate CD68 immunoreactivity in the irradiated brain (9 Gy+PMX205), indicating prevention of inflammation. (D-E) Volumetric quantification for the microglial C5aR1 expression (C5aR1-IBA1 immunoreactivity, magenta-green) showed a radiation-induced increase in the microglial-C5aR1 in the WT hippocampal dg and dh compared to unirradiated controls. PMX205 treatment reduced microglial-C5aR1 expression compared to the 9 Gy mice +vehicle ( F ). (G) Analysis of astrogliosis (GFAP-C3d) showed radiation-induced elevation in GFAP + astrocytic surface co-labeled with complement C3d (GFAP surface rendering, magenta, and C3d spot rendering, green) in the 9 Gy+Vehicle compared to 0 Gy+Vehicle or 0 Gy+PMX205 groups in the hippocampal dg . 9 Gy mice receiving C5aR inhibitor (PMX205) did not show elevated GFAP-C3d immunoreactivity, indicating reduced astrogliosis (H) . Mean±SEM ( N =6-8 per group). Scale bars, 50 µm ( A-B, D-E ) and 10 µm ( G ).

Article Snippet: This included 3% normal goat serum, NGS with 1% Bovine Serum Albumin or BSA in Tris-A buffer for synaptophysin; 4% BSA in Tris-A buffer for PSD95; 3% NGS in Tris-A buffer for CD88 (C5aR1) and CD68/IBA1 markers (NGS, Jackson ImmunoResearch Cat. 005-000-121; BSA, Sigma-Aldrich Cat. 05470) at rt for 45 minutes.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Activation Assay, Irradiation, Expressing, Inhibition, Labeling