Journal: Frontiers in Immunology

Article Title: Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Suppresses Decidual M1 Macrophage Polarization Against Inflammation in Lipopolysaccharide-Induced Preeclampsia-Like Mouse Model

doi: 10.3389/fimmu.2021.642071

Figure Lengend Snippet: A higher expression of CD86 on decidual macrophages (dMφ) with a lower expression of CD163 on dMφ was found in PE patients. (A) Representative immunofluorescence images of CD86 (M1 biomarkers) on dMφ in PE patients and normal controls; the relative CD86 positive CD68 immunoreactivity was quantified as shown in the graph. CD68 was stained as pan-macrophage biomarker. White arrows indicated positive stained signals. (B) Representative immunofluorescence images of CD163 (M2 biomarkers) on dMφ in PE patients and normal controls; quantification for the relative CD163 positive dMφ immunoreactivity was shown in the graph. White arrows indicated positive stained signals. Scale bar=30 μm. * P < 0.05.

Article Snippet: Primary antibodies incubation was processed at 4°C for 20 hrs, the information for single immunofluorescent staining was as following: anti-MMP-9 (rabbit, 1:100, cat#AF5228, Affinity Biosciences, Cincinnati, OH, USA), anti-α-SMA (rabbit, 1:100, cat#bs-10196R, Bioss Biotechnology, Beijing, China); the information for double immunofluorescent staining was as following: α7nAChR (rabbit, 1:50, cat# ab10096, Abcam, Cambridge, MA, USA), CD68 (mouse,1:50, cat# ab955), CD86 (rabbit, 1:50, cat# bs1035R, Bioss Biotechnology), CD163(rabbit, 1:50, cat# ab182422), anti-E-cadherin (mouse, 1:40, cat#sc-8426, Santa Cruz Biotechnology, Dallas, TX, U.S.A.), anti-cytokeratin 7 (CK7, rabbit, 1:20, cat# bs-1744R, Bioss Biotechnology).Sections were then rinsed in PBS and incubated with a secondary antibody for 3 hrs at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Biomarker Assay

Journal: Frontiers in Immunology

Article Title: Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Suppresses Decidual M1 Macrophage Polarization Against Inflammation in Lipopolysaccharide-Induced Preeclampsia-Like Mouse Model

doi: 10.3389/fimmu.2021.642071

Figure Lengend Snippet: Nicotine upregulated the decreased α7nAChR activity (CHAT expression) on dMφ from LPS-induced PE-like mice. (A, B) Percentages of CHAT + dMφ in all experimental groups were calculated by flow cytometric analysis. Total leukocytes were gated using FSC vs. SSC and then gated for identifying CD68 + macrophages. Representative data were derived from separate mice in each group. Data are presented as means ± SEM of 10 pregnant mice per group. * P < 0.05.

Article Snippet: Primary antibodies incubation was processed at 4°C for 20 hrs, the information for single immunofluorescent staining was as following: anti-MMP-9 (rabbit, 1:100, cat#AF5228, Affinity Biosciences, Cincinnati, OH, USA), anti-α-SMA (rabbit, 1:100, cat#bs-10196R, Bioss Biotechnology, Beijing, China); the information for double immunofluorescent staining was as following: α7nAChR (rabbit, 1:50, cat# ab10096, Abcam, Cambridge, MA, USA), CD68 (mouse,1:50, cat# ab955), CD86 (rabbit, 1:50, cat# bs1035R, Bioss Biotechnology), CD163(rabbit, 1:50, cat# ab182422), anti-E-cadherin (mouse, 1:40, cat#sc-8426, Santa Cruz Biotechnology, Dallas, TX, U.S.A.), anti-cytokeratin 7 (CK7, rabbit, 1:20, cat# bs-1744R, Bioss Biotechnology).Sections were then rinsed in PBS and incubated with a secondary antibody for 3 hrs at room temperature.

Techniques: Activity Assay, Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Suppresses Decidual M1 Macrophage Polarization Against Inflammation in Lipopolysaccharide-Induced Preeclampsia-Like Mouse Model

doi: 10.3389/fimmu.2021.642071

Figure Lengend Snippet: Immunofluorescent staining results showed that nicotine differentially affected the expression of decidual macrophage M1 and M2 biomarkers in PE-like mice. (A) Immunostaining of CD86 and CD68 in the decidua from all animal groups and bar graph showed quantitative analysis results on CD86-positive immunoreactivity in dMφ. White arrow heads showed colocalization of dMφ with CD86. Scale bar=30 μm. ** P < 0.01. (B) Immunostaining of CD163 and CD68 in the decidua from all animal groups and bar graph showed quantitative analysis results on CD163-positive immunoreactivity in dMφ. White arrow heads showed colocalization of dMφ with CD163. Scale bar=30 μm. * P < 0.05 and ** P < 0.01.

Article Snippet: Primary antibodies incubation was processed at 4°C for 20 hrs, the information for single immunofluorescent staining was as following: anti-MMP-9 (rabbit, 1:100, cat#AF5228, Affinity Biosciences, Cincinnati, OH, USA), anti-α-SMA (rabbit, 1:100, cat#bs-10196R, Bioss Biotechnology, Beijing, China); the information for double immunofluorescent staining was as following: α7nAChR (rabbit, 1:50, cat# ab10096, Abcam, Cambridge, MA, USA), CD68 (mouse,1:50, cat# ab955), CD86 (rabbit, 1:50, cat# bs1035R, Bioss Biotechnology), CD163(rabbit, 1:50, cat# ab182422), anti-E-cadherin (mouse, 1:40, cat#sc-8426, Santa Cruz Biotechnology, Dallas, TX, U.S.A.), anti-cytokeratin 7 (CK7, rabbit, 1:20, cat# bs-1744R, Bioss Biotechnology).Sections were then rinsed in PBS and incubated with a secondary antibody for 3 hrs at room temperature.

Techniques: Staining, Expressing, Immunostaining

Journal: Frontiers in Immunology

Article Title: Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Suppresses Decidual M1 Macrophage Polarization Against Inflammation in Lipopolysaccharide-Induced Preeclampsia-Like Mouse Model

doi: 10.3389/fimmu.2021.642071

Figure Lengend Snippet: FCM analysis results showed that nicotine prompted the polarization of M1 dMφ to M2 dMφ from LPS-induced PE-like mice. (A) FCM analysis of the percentage of CD86 + , TNF-α + , IL-1β + and iNOS + dMφ from different animal groups (n=10 each). Total leukocytes were gated using FSC vs. SSC and then gated for identifying CD68 + macrophages. Gating strategy was the same to that used to identify CD68 + CHAT + cells. (B) FCM analysis of the percentage of CD206 + , CD 163 + , IL-10 + and Arg-1 + dMφ from different animal groups (n=10 each). * P < 0.05 and ** P < 0.01.

Article Snippet: Primary antibodies incubation was processed at 4°C for 20 hrs, the information for single immunofluorescent staining was as following: anti-MMP-9 (rabbit, 1:100, cat#AF5228, Affinity Biosciences, Cincinnati, OH, USA), anti-α-SMA (rabbit, 1:100, cat#bs-10196R, Bioss Biotechnology, Beijing, China); the information for double immunofluorescent staining was as following: α7nAChR (rabbit, 1:50, cat# ab10096, Abcam, Cambridge, MA, USA), CD68 (mouse,1:50, cat# ab955), CD86 (rabbit, 1:50, cat# bs1035R, Bioss Biotechnology), CD163(rabbit, 1:50, cat# ab182422), anti-E-cadherin (mouse, 1:40, cat#sc-8426, Santa Cruz Biotechnology, Dallas, TX, U.S.A.), anti-cytokeratin 7 (CK7, rabbit, 1:20, cat# bs-1744R, Bioss Biotechnology).Sections were then rinsed in PBS and incubated with a secondary antibody for 3 hrs at room temperature.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage

doi: 10.1172/JCI95612

Figure Lengend Snippet: (A) Left: Representative flow cytometry plots of TER119 and CD45 expression from brains at days 1 and 3 after ICH. Population TER119+CD45– and TER119–CD45+ quadrants representing eryptotic erythrocytes and apoptotic leukocytes, respectively, and their percentages are shown. Right: Quantification of absolute number of PtdSer-positive RBCs and WBCs in the brains at days 1 and 3 after ICH. n = 3/group. *P < 0.05 versus day 1 group by Student’s t test. (B) Representative flow cytometry shows LIVE/DEAD–annexin V+ population from non–heat-shocked erythrocytes (0 minutes) and 56°C heat-shocked (HS) erythrocytes (5 minutes), and their percentages are shown. n = 3/group. (C) Top: Representative immunofluorescence images show engulfment of PHK-26–labeled HS erythrocytes (red) in normal and thrombin-stimulated CD11b-positive (green) BMDMs with or without annexin V incubation, with higher magnification of the boxed area in the inset. Bottom: Quantification of n = 3/group; each independent experiment includes 2 technical replicates. *P < 0.05 versus control+HS group; #P < 0.05 versus thrombin+HS group by Student’s t test. (D) Gene expression for markers of proinflammatory (Tnf and Cd86) and reparative (Hmox1 and Clec7a) phenotypes from BMDMs with or without HS treatment under normal or thrombin-stimulated conditions. n = 3/group. *P < 0.05 versus thrombin group by 1-way ANOVA and Bonferroni’s post hoc test. (E) Representative phase contrast images showing control and thrombin-stimulated BMDMs treated with HS and beads, with inset of higher magnification of the boxed area showing engulfment. n = 3/group. (F) Gene expression of Tnf and Cd86 in thrombin-stimulated BMDMs is reduced after HS but not bead treatment. n = 3/group. *P < 0.05 versus control group; #P < 0.05 versus thrombin group by Student’s t test. An.V, annexin V; B, beads; C, control; T, thrombin.

Article Snippet: The primers and probes for mouse Tnf (TaqMan Gene Expression Assay ID Mm00443258_m1), Cd86 (Mm01344642_m1), Hmox1 (Mm00516005_m1), and Clec7a (Mm01183349_m1), as well as human TNF (HS01113624_g1) and HMOX1 (HS01110250_m1), were obtained from Thermo Fisher Scientific.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Labeling, Incubation, Control, Gene Expression

Journal: Theranostics

Article Title: Non-invasive photobiomodulation treatment in an Alzheimer Disease-like transgenic rat model

doi: 10.7150/thno.70756

Figure Lengend Snippet: PBM treatment promotes microglial polarization from M1 to M2 phenotype. (A) Representative confocal microscopy images and 3D reconstruction images of Iba-1 with M1 marker CD16/32 or the M2 marker CD 206 in both the cortex and hippocampus. The relative fluorescent intensities of CD16/32 and CD206 were analyzed using Image J. Data are presented as mean ± SEM (n = 5). Rectangles indicate cells enlarged and 3D-rendered in the bottom row. Scale bar = 20 µm. (B) Western blotting analysis of M1 phenotype markers (i.e., CD32, CD86, and iNOS) and M2 phenotype markers (i.e., TGFβ and ARG). Data are presented as mean ± SEM (n = 4). (C) Immunofluorescence staining of Iba-1 with M1 marker CD16/32 or the M2 marker CD 206 in vitro cell culture. Scale bar = 20 µm. Data are presented as mean ± SEM (n = 6). * P < 0.05 versus WT group, # P < 0.05 versus AD or Aβ1-42 group.

Article Snippet: After incubation with 3% bovine serum albumin (BSA) for 30 min, the membranes were then incubated at 4 °C overnight with the following antibodies: Bax (Abcam), PSD95 (Thermo Fisher), spinophilin (Abcam), synaptophysin (Abcam), GAPDH (Abcam), CD86 (Proteintech), iNOS (Abcam), CD32 (Proteintech), TGFβ (Proteintech), ARG (Proteintech), MFF (Proteintech), FIS1 (Proteintech), Drp1 (BD Biosciences), MFN2 (Proteintech), OPA1 (BD Biosciences), COX4 (Proteintech), Hbα (Abcam), Bcl-2 (Santa Cruz Biotechnology), and β-actin (Proteintech).

Techniques: Confocal Microscopy, Marker, Western Blot, Immunofluorescence, Staining, In Vitro, Cell Culture

Journal: Antioxidants

Article Title: Genipin Attenuates Diabetic Cognitive Impairment by Reducing Lipid Accumulation and Promoting Mitochondrial Fusion via FABP4/Mfn1 Signaling in Microglia

doi: 10.3390/antiox12010074

Figure Lengend Snippet: GEN shifted microglia polarization and inhibited lipid accumulation in LPS/HG/PA-induced HMC3 cells. The HMC3 cells were treated with GEN (5, 10, 20 μM) for 4 h and then stimulated with LPS/HG/PA for 12 h. The supernatant concentrations of TNF-α ( A ), IL-6 ( B ), IL-4 ( C ) and IL-10 ( D ) were examined by ELISA (n = 4). The mRNA expressions of iNOS ( E ), CCL2 ( F ), ARG1 ( G ) and YM1 ( H ) in cells were assessed by PCR (n = 4). The population of CD68-positive and CD206-positive cells were measured by flow cytometry ( I ). The expressions of FABP4, p-NF-κB and NF-κB were detected by Western blot ( J – L ) (n = 3). The nucleus translocation of NF-κB was visualized by immunofluorescence staining under laser confocal microscope. The scale bar equaled 5 μm ( M ). The lipid accumulation was observed by oil red O staining. The scale bar equaled 50 μm ( N ). The analysis of oil red O staining ( O ). The mRNA expressions of fatty acid β-oxidation genes including ACOX1 , ACAA2 and ECHS1 were measured by PCR ( P ). The mRNA expressions of fatty acid uptake genes including SLC27A1 and PPARα were measured by PCR ( Q ). The mRNA expressions of fatty acid synthesis genes including FASN and ACLY were measured by PCR ( R ). The results are expressed as means ± SDs. ### p < 0.001 compared with control group. * p < 0.05, ** p < 0.01 compared with LPS/HG/PA group or the other group. ns means not significant.

Article Snippet: Mfn2 (#9482S), Drp1 (#8570S), p-Nuclear Factor Kappa B (NF-κB) (#3033), NF-κB (#8242), CD68 (#91882), p47phox (#4312), β-actin (#4970), Na, K-ATPase (#3010), ubiquitin (#3936), Flag (#8146), Flag (#14793), HA (#3724), Myc (#2276), V5 (#80076), mouse anti-rabbit IgG (Conformation Specific, #5127), rabbit anti-mouse IgG (Light Chain Specific, #58802) and mouse anti-rabbit IgG (Light-Chain Specific, #93702) antibodies were provided by Cell Signaling Technology (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot, Translocation Assay, Immunofluorescence, Staining, Microscopy, Control

Journal: Nature Communications

Article Title: Prussian blue nanoparticles targeting multiple PANoptosome-mediated PANoptosis for myocardial ischemia-reperfusion injury therapy

doi: 10.1038/s41467-026-70012-2

Figure Lengend Snippet: A–E Echocardiographic analysis of left ventricular ejection fraction (LVEF), LV fraction shortening (LVFS), LV end-systolic volume (LVESV), and LV end-diastolic volume (LVEDV) on preoperative day 1 and postoperative days 1, 14, and 28. Scale bar: 100 ms. Exact P- values from upper to lower: ( B ) day 14 (0.0012), day 28 (0.00003, 0.0028); ( C ) day 14 (0.0056), day 28 (0.0002, 0.039); ( D ) day 14 (0.0011, 0.0201), day 28 (0.00001, 0.0004); ( E ) day 14 (0.015, 0.0294), day 28 (0.0101). F Representative Masson and HE staining in hearts on 28 days post-surgery. Scale bar: 1 mm. G Fibrosis quantification in hearts 28 days post-surgery. Exact P- values from upper to lower: 2.28E-08, 3.28E-07, 0.0116. H Evans blue/TTC staining and quantitative analysis showing the cardiac cell survival and blood perfusion in different groups on day 3 post-surgery. AAR: area at risk. IF: infarct zones. Scale bar: 1 mm. Exact P- values from upper to lower: (AAR/LV) 0.4990 for MIRI vs. PB, 0.3119 for MIRI vs. PB@PM, 0.9269 for PB vs. PB@PM; (IF/AAR) 3.48E-08, 0.0001, 0.00002. I , J TUNEL (red) and cTnT (green) co-staining and quantification in peri-infarct zones (day 3). Scale bar: 50 μm. Exact P- values from upper to lower: 0.000005, 0.0006, 0.0154. K , L Representative immunofluorescence images and quantitative analysis of CD86 (red) in peri-infarct regions (day 3). Scale bar: 1 mm or 50 μm. Exact P- values from upper to lower: 2.17E-08, 0.00001, 0.00005. M , N Representative immunofluorescence staining and quantitative analysis of Ly-6G (red) in peri-infarct regions (day 3). Scale bar: 1 mm or 50 μm. Exact P- values from upper to lower: 8.27E-09, 5.89E-08, 9.99E-07. O–R Representative immunofluorescence staining and quantification analysis of α-SMA (red)/cTnT (green) and CD31(red)/cTnT (green) in peri-infarct region (day 28). Scale bar: 1 mm or 50 μm. Exact P- values from upper to lower: ( P ) 0.000002, 0.0005, 0.0026; ( R ) 0.000004, 0.0003, 0.016. Data: mean ± SD of 5 biologically independent replicates. Significance: one-way ANOVA with Tukey’s post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001; ns, P > 0.05). Source data are provided as a Source Data file.

Article Snippet: CD86 (19589 s), Bax (5023 T), ASC (40002 T), caspase 3 (9662S), cleaved caspase 3 (9664 T), cleaved GSDMD (10137 T), TLR4 (14358S), NF-κB p65/RelA (8242 T), phospho-NF-κB p65/RelA (3033 T), phospho-RIPK3 (91702 T), and GAPDH (2118 T) antibodies were purchased from Cell Signaling Technology, USA.

Techniques: Staining, TUNEL Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: Pathology-inspired collagen-binding thermosensitive micelle drops enable prolonged and efficient treatment of fungal keratitis

doi: 10.1016/j.bioactmat.2025.04.011

Figure Lengend Snippet: Therapeutic mechanism of PX-TA-AmB in FK via MAPK6/PI3K/AKT pathway modulation. (A) Volcano plot of the differentially expressed genes. (B) KEGG pathway enrichment analysis of the differentially expressed genes. (C) Heatmap of the differentially expressed genes related to the inflammatory response, signaling pathway regulation, and corneal scarring before and after PX-TA-AmB treatment. (D, E) GSEA with the indicated KEGG and Reactome gene sets. (F, G, H) MAPK6, α-SMA, and LOX mRNA expression levels in treated versus control corneal tissues. (I) Representative Western blot images of p-AKT, AKT, p-PI3K, PI3K, MAPK6, Vinculin, and β-actin proteins in different treatment groups. (J) Representative Western blot images of IL-1β and MMP9 in different treatment groups. (K) Representative Western blot images for functional validation of the role of AKT pathway in PX-TA-AmB-mediated downregulation of IL-1β and MMP9 using the AKT activator SC79. (L) Immunofluorescence assessment of α-SMA, MMP9, CD206, CD86, and Ly6G under different treatments. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. Mean ± SD, n = 3; one-way ANOVA.

Article Snippet: The primary antibodies used included Ly6g (eBioscience, 14–5931, 1:50), CD86 (Proteintech, 13395-1-AP, 1:150), CD206 (Proteintech, 18704-1-AP, 1:150), MMP9 (Affinity Biosciences, AF5228, 1:200), α-SMA (Proteintech, 14395-1-AP, 1:150), IL-6 (Affinity Biosciences, DF6087, 1:200), and IL-1β (Affinity Biosciences, AF5103, 1:200).

Techniques: Expressing, Control, Western Blot, Functional Assay, Biomarker Discovery, Immunofluorescence

Journal: Skeletal Muscle

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

doi: 10.1186/s13395-026-00417-6

Figure Lengend Snippet: TRPV1 activation leads to a difference in macrophage infiltration with a remarkable increase of M1 and a decrease of M2 in number in vivo. A Representative immunofluorescence images of F4/80 after treatment with CAP and CPZ in CTX-induced muscle tissue at 4d. B The percentage of F4/80-positive cells between four groups at 4d post-injury ( n = 5 animals per experimental group; mean ± SD; One-way ANOVA). C , D Representative immunofluorescence images showed the relative fluorescence intensity of TRPV1 in these F4/80 + macrophages in each group ( n = 5 animals per experimental group; mean ± SD; One-way ANOVA). E , F Representative immunofluorescence images showed the infiltration of numbers of M1 (F4/80 + and CD86+) and M2 (F4/80 + and CD206+) macrophages in different groups at different intervals. G , H The proportion of M1 (F4/80 + and CD86+) macrophages and M2 macrophages (F4/80 + and CD206+) between four groups at 2-8days post-injury ( n = 5 animals per experimental group; mean ± SD; Two-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Article Snippet: Macrophage subtypes (M1: CD86-positive and F4/80-positive; M2: CD206-positive and F4/80-positive) were counted independently by two pathologists in five randomly selected non-overlapping fields per section at 400× magnification using a fluorescence microscopy (ECLIPSE Ti2, Nikon, Japan).

Techniques: Activation Assay, In Vivo, Immunofluorescence, Fluorescence

Journal: Skeletal Muscle

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

doi: 10.1186/s13395-026-00417-6

Figure Lengend Snippet: TRPV1 regulates M1/M2 macrophage polarization to promote myogenic differentiation in C2C12 cells. A C2C12 myoblasts were co-cultured with M1 or M2 macrophages for 4 days via a transwell cell culture insert. B Representative Western blot bands of MyoD and MYH3 in C2C12 myoblasts after being co-cultured with M1 or M2 macrophages for 4 days. C , D The protein expression of MyoD and MYH3 in those C2C12 myoblasts which were co-cultured with M1or M2 macrophages after CAP and CPZ treatment ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). E , F Representative immunofluorescence images showed myotubes fusion index in C2C12 cells ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Article Snippet: Macrophage subtypes (M1: CD86-positive and F4/80-positive; M2: CD206-positive and F4/80-positive) were counted independently by two pathologists in five randomly selected non-overlapping fields per section at 400× magnification using a fluorescence microscopy (ECLIPSE Ti2, Nikon, Japan).

Techniques: Cell Characterization, Cell Culture, Western Blot, Expressing, Immunofluorescence

Journal: Bioactive Materials

Article Title: A self-locking conductive cardiac patch for immediate electrical integration with infarcted rat myocardium

doi: 10.1016/j.bioactmat.2025.10.045

Figure Lengend Snippet: The effects of patches on angiogenesis and inflammation in infarcted tissues 4 weeks post-MI. a) Immunofluorescence staining of α-smooth muscle actin (α-SMA, a vascular protein marker, green) and von Willebrand factor (vWF, an endothelial marker, red) of the infarct region in the MI, BMN-P, BMN-CP and CBMN-CP groups. b, c) Quantitative analysis of α-SMA (b) and vWF (c) in different groups based on fluorescent staining images (n = 4). d) Representative immunofluorescence staining of the infarct region in different groups for CD86 (green) and CD206 (red). e, f) Fluorescence intensity statistics of CD86 (e) and CD206 (f) in different groups based on fluorescent staining images (n = 4). Nuclei are stained blue with DAPI. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The tissue sections were then incubated with the following primary antibodies: rabbit Connexin 43 primary antibody (BOSTER, BA1727, 1:200, China), mouse α-actinin primary antibody (Abcam, AB9465, 1:200, UK), mouse α-SMA primary antibody (Wuhan Sanying, 67735-1-IG, 1:400, China), rabbit vWF primary antibody (Wuhan Sanying, 27186-1-AP, 1:300, China), mouse CD86 primary antibody (BOSTER, BA4121, 1:100, China) and rabbit CD206 primary antibody (Wuhan Sanying, 18704-1-AP, 1:400, China).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence

Journal: eLife

Article Title: Metabolic and non-metabolic liver zonation is established non-synchronously and requires sinusoidal Wnts

doi: 10.7554/eLife.46206

Figure Lengend Snippet: ( A ) Experimental strategy for Cldn2-EGFP mice injected with CCl 4 . ( B ) ( No CCl 4 ) Results of double-immunofluorescence analysis show expression of the pan-macrophage marker F4/80 + in Kupffer cells (arrowheads) in a control adult liver (no CCl 4 injection). ( Day 2–5 ) In the CCl 4 -injected Cldn2-EGFP liver, macrophages (arrows) start to infiltrate the necrotic perivenous area at day two and form a physical barrier between the central vein endothelium and the expanding GFP + Zone 2 at days 3 and 5. Resolution of macrophage perivenous infiltrates and restoration of claudin-2/GFP expression occur 7 days post-CCl 4 injection (arrows are Kupffer cells). ( C ) ( No CCl 4 ) M1 macrophages (CD86 + ) are not detected in perivenous or periportal regions in Cldn2-EGFP control mice ( C is central vein, P is portal vein). ( Day 2–5, top ) Immunofluorescence images of CCl 4 -injected Cldn2-EGFP livers showing increasingly abundant infiltrates of CD86+ macrophages (arrows) 2 days post-CCl 4 and persistence of these cells around the central veins at days 3 and 5. CD86 + macrophages are no longer present in perivenous areas at day 7 ( CV , central vein region). ( Day 2–5, bottom ) CD86 + macrophages are very scarce in periportal areas in CCl 4 -injected livers ( PV , portal vein region). (The exposure in the green channel was decreased for better visualization of the red [CD86] signal. ( D ) Macrophages infiltrating the perivenous region do not express the M2 macrophage marker CD206 3 days post-CCl4 injection (arrowheads indicate low expression of this marker in LSECs). ( E ) Quantification of F4/80 + immunofluorescence distribution 2-, 3- and 5 days post-CCl 4 . p values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test. **p<0.01, ***p<0.001. 3–4 representative fields from three individual livers dissected at the indicated time points were used for quantification. Asterisks and C , central veins. P, portal veins. CV , central vein area. PV , portal vein area. ( F ) A few CD86 + macrophages are seen within the Lyve1 + hepatic sinusoids in periportal areas of the CCl 4 injected liver. CD86 + macrophages are overabundant in perivenous areas (right, ‘C’, arrowhead) in comparison to periportal areas (‘P’). Each image represents 3–4 individual livers. Scale bars: 100 μm ( B ), ( C ) [no CCl 4 , Days 2,7]) Scale bars: 50 μm ( C ) [Days 3,5], ( D,F ). Figure 5—source data 1. Quantification of F4/80 + immunofluorescence distribution post-CCl 4 injection.

Article Snippet: Antibody , anti-mouse CD86 (rat monoclonal) , SouthernBiotech , Cat. #: 1735–01; RRID: AB_2795211 , IF (1:100).

Techniques: Injection, Immunofluorescence, Expressing, Marker, Control, Comparison

Journal: eLife

Article Title: Metabolic and non-metabolic liver zonation is established non-synchronously and requires sinusoidal Wnts

doi: 10.7554/eLife.46206

Figure Lengend Snippet:

Article Snippet: Antibody , anti-mouse CD86 (rat monoclonal) , SouthernBiotech , Cat. #: 1735–01; RRID: AB_2795211 , IF (1:100).

Techniques: Imaging, cDNA Synthesis, Activity Assay, Plasmid Preparation, Western Blot, RNAscope, Positive Control, Negative Control, Sequencing, Cell Culture, Protease Inhibitor, Recombinant, Mutagenesis, Software