Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: Astrocytic IL-33 is robustly upregulated after hypoxic-ischemia in neonatal mouse brains. a Western blot of IL-33 was performed using protein extracted from the mouse brain at 1, 3, 7, and 14 days after HI or sham operation. Data are mean ± SEM ( n = 6 in each group). # P < 0.01 compared to sham. b Representative images of IL-33 (green) and GFAP or Olig2 (red) labeling in the cerebral cortex of WT mice at 1 day after HI injury. No IL-33 staining was observed in Neun or Iba1 cells 1 d after HI. Scale bar, 25 μm. Nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI). c Semi-quantitative analysis of glia or neuron-type cell contributions to the IL-33-positive cell population in vehicle group 1 day after HI. Data are mean ± SEM ( n = 6 in each group). * P < 0.05 compared to sham. d Representative IL-33 expression on astrocytes in WT mice at 1 day after HI injury. The numbers shown indicate the percentage of IL-33 expression on the indicated cell population. Data are mean ± SEM ( n = 6 in each group). * P < 0.05 compared to sham

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: Western Blot, Labeling, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: ST2 expression on astrocytes is elevated after HI. a Histograms showing ST2 expression on astrocytes, oligodendrocytes, microglia, and neurons in the brain 3 days after HI or sham operation. The blue and red lines represent staining in sham and HI brains, respectively, at 3 days after surgery. The gray area represents isotype control staining. b–e Percentages of ST2 expression on astrocytes ( b ), oligodendrocytes ( c ), microglia ( d ), and neurons ( e ) in sham brain and HI brain at 3 days after surgery. Data are mean ± SEM ( n = 5–7 per group), * P < 0.05 compared to sham. f Representative images of GFAP (green) and ST2 (red) labeling in the cerebral cortex 3 days after HI. Scale bar, 25 μm. Nuclei were stained blue with DAPI

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: Expressing, Staining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Neuroprotective effect of astrocyte-derived IL-33 in neonatal hypoxic-ischemic brain injury

doi: 10.1186/s12974-020-01932-z

Figure Lengend Snippet: IL-33 promotes astrocyte survival and proliferation after HI injury. a Representative FACS analysis of ST2 + astrocytes at 24 h after 6 h OGD. Right, numbers indicate the percentage of ST2 + astrocytes at 24 h after 6 h OGD. Data are mean ± SEM ( n = 3 in each group). * P < 0.05 compared to untreated controls. b CCK-8 assay in astrocyte-enriched cultures subjected to 6 h OGD or sham conditions followed by treatment with PBS or a range of concentrations of IL-33 for another 24 h. Data are mean ± SEM ( n = 3 in each group). # P < 0.01 compared to untreated controls. c Representative images of Ki-67 (red) and GFAP (green) in the cerebral cortex of IL-33- and PBS-treated mice at 7 days post HI (left). Scale bar, 25 μm. Right, quantification of Ki-67 and GFAP dual-labeled cells at 7 days post HI ( n = 9 per group). d , e Percentages of phosphorylated AKT (pAKT) ( d ) and Ki-67 + ( e ) in astrocytes at 24 h after 6 h OGD and the culture conditions shown. Data are mean ± SEM ( n = 3 in each group). * P < 0.05, # P < 0.01. f Representative FACS analysis of cell-cycle status of astrocytes at 24 h after 6 h OGD and the culture conditions shown. Data are mean ± SEM ( n = 3 in each group). * P < 0.05

Article Snippet: The sections were stained overnight at 4 °C with either anti-ST2 (1:200, ab25877, Abcam), anti-IL-33 (1:500, AF3626, R&D), anti-GFAP (1:500, 104805-T08, Sino Biological Inc.), anti-Olig2 (1:500, EPR2673, Abcam), anti-Iba1(1:50, 20A12.1, Sigma-Aldrich), anti-Neun (1:50, A60, Sigma-Aldrich), or anti-Ki67 (1:500, ab15580, Abcam) followed by PE- or FITC-conjugated secondary antibodies (eBioscience).

Techniques: CCK-8 Assay, Labeling

Journal: Gene therapy

Article Title: Targeted inhibition of platelet-derived growth factor receptor-beta subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats.

doi: 10.1038/gt.2008.93

Figure Lengend Snippet: Figure 3 Localization of LacZ driven by CMV promoter and GFAP promoter in normal rats, acute liver injury rats and chronic liver injury rats. (a) Representative graphs of b-galactosidase (b-gal) immunofluorescence showed the distribution of b-gal-positive cells in normal rats, acute liver injury rats and chronic liver injury rats treated with pCMV-shRNA-LacZ or pGfa-shRNA-LacZ. Magnification of 20. The scale bar represents 80 mm. (b) Representative graphs of a-SMA and GFAP immunofluorescence showed that in the chronic injured liver, a-SMA and GFAP proteins distributed mainly around the portal area and the hyperplastic bile duct, whereas in the acute injured liver, both the proteins revealed a much more diffuse distribution in the hepatic lobule. In addition, the GFAP-positive cells were more than the a-SMA- positive cells both in the acute injured liver and in the chronic injured liver. Magnification of 20. The scale bar represents 80 mm. (c) Representative graphs of b-gal and a-SMA double-staining revealed that b-gal protein in pCMV-shRNA-LacZ-treated livers could be expressed in hepatocytes (blue arrow) and activated HSCs stained by a-SMA (white arrow), magnification of 63. The scale bar represents 40 mm. (d) Representative graphs of b-gal and GFAP double-staining showed that b-gal-positive cells were all GFAP-positive HSCs (pink arrow) in pGfa-shRNA-LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. (e) Representative graphs of b-gal and a-SMA double-staining showed that some b-gal protein was expressed in activated HSCs stained by a-SMA (white arrow) in pGfa-shRNA- LacZ-treated livers, magnification of 63. The scale bar represents 40 mm. CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; HSCs, hepatic stellate cells; PDGFR-b, platelet-derived growth factor receptor-b subunit; a-SMA, a-smooth muscle actin; shRNA, short hairpin RNA; RT-PCR, reverse transcriptional-PCR.

Article Snippet: Frozen liver sections (8 mm in thickness) were incubated with mouse anti-b-galactosidase monoclonal antibody (Promega, Madison, WI, USA), rabbit anti-GFAP polyclonal antibody (Boster, Wuhan, China) or rabbit anti-a-SMA polyclonal antibody (Bios, Beijing, China) for 20 h at 4 1C, then washed in phosphate-buffered saline and incubated for 30 min with Cy5-labeled anti-rabbit IgG antiserum and FITC-labeled anti-mouse IgG antiserum (Jackson, West Grove, PA, USA) at 1:100 dilution in phosphate-buffered saline.

Techniques: shRNA, Double Staining, Staining, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization

doi: 10.1177/09636897231163232

Figure Lengend Snippet: Antibodies Used for Flow Cytometry and Immunofluorescence Staining.

Article Snippet: TA302094 , Human GFAP , Origene.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Plasmid Preparation

Journal: Cell Transplantation

Article Title: Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization

doi: 10.1177/09636897231163232

Figure Lengend Snippet: Long-term (6 months) engraftment and neuronal and glial differentiation of hiPSC-NPCs after grafting into striata of the immunodeficient rat. (A) IF analysis of the coronal brain section showed a consistent presence of hNUMA+ grafts in the targeted striatal region. Staining with human-specific GFAP shows the homogenous distribution of grafted NPC-derived astrocytes throughout the graft. (B) Intense human-specific NSE expression (green area) with well morphologically defined human astrocytes (red signal). (C) Areas occupied with mature grafted human neurons (hNSE) show intense human-specific synaptophysin (hSYN) expression. (D) hSYN expression is primarily present in areas with a high density of grafted neuron-derived human axons (HO14+), (yellow arrows). (E) Expression of mature neuronal marker NeuN in hNUMA+ grafted neurons. (F) A high density of VGAT punctate-like expression in areas of hSYN immunoreactivity. hSYN+ terminals show co-localization with VGAT immunoreactivity and are opposed to post-synaptic gephyrin immunoreactivity (yellow arrows). (G) Presence of vimentin+ glial progenitors at the periphery of hNUMA+ graft. (H) Expression of Olig2 (oligodendrocyte marker) immunoreactivity in hNUMA+ grafted cells. (I) Hematoxylin-eosin-stained section with normally appearing graft. No hyper-cellularity (indicative of tumor formation) can be identified (I-1, I-2). ( J ) Quantitative analysis of neuronal and glial marker(s) expression. Scale bars: 20 µm (A–H), 10 µm (A–H; inserts). *P < 0.05; * *P < 0.01, all data are presented as mean ± SEM, n = 3, two-tailed t test. DCX: doublecortin; GFAP: glial fibrillary acidic protein; hiPSCs-NPC: human induced pluripotent stem cells–derived neural precursors; hNUMA: human-specific nuclear marker; IF: immunofluorescence; NeuN: DNA-binding neuron-specific protein; SEM: standard error of the mean; VGAT: vesicular GABA transporter.

Article Snippet: TA302094 , Human GFAP , Origene.

Techniques: Staining, Derivative Assay, Expressing, Marker, Two Tailed Test, Immunofluorescence, Binding Assay

Journal: Brain, behavior, and immunity

Article Title: Chemotherapy accelerates age-related development of tauopathy and results in loss of synaptic integrity and cognitive impairment

doi: 10.1016/j.bbi.2019.04.005

Figure Lengend Snippet: (A and B): Brains of control and cisplatin treated mice were co-stained for Tau-1 and GFAP. Representative examples of GFAP staining in an area of minimal Tau-1 clusters in control mice (A) and overt Tau-1 pathology in cisplatin treated mice (B). (Top row; 40x objective; Bottom row; 63x objective). The higher magnification demonstrates that there is no overlap between Tau-1 clusters and GFAP. C. GFAP staining in areas without Tau-1 clusters (left and middle panel) and control staining with secondary antibodies only (right). C. Double immunofluorescence analysis of Tau-1 and Iba-1 in brain of control (top) and cisplatin-treated (bottom) mice in areas of Tau-1 clusters. D. GFAP (top) and Iba-1 (bottom) expression in brain areas without Tau-1 clusters in control (left) and cisplatintreated mice (middle) and control with secondary antibodies only (right).

Article Snippet: Slides were incubated with the following antibodies: mouse anti-Tau-1 (1:200, Millipore), mouse anti-phospho-Tau (AT8) (1:100, Thermo Scientific), rabbit anti-GFAP (1:1000, Acris Antibodies), rabbit anti-Iba-1 (1:500, Wako), rabbit anti-synaptophysin (1:1000, Millipore), rabbit anti-PSD95 (1:1000, Abcam), followed by secondary antibody incubation in Alexa-488 goat-anti-rabbit (1:1000, Invitrogen, Grand Island, NY) for synaptophysin, Alexa-488 goat anti-mouse (1:500, Invitrogen, Grand Island, NY) for Tau-1 and AT8, and Alexa-594 goat anti-rabbit (1:500, Invitrogen, Grand Island, NY) for GFAP, Iba-1, and PSD95.

Techniques: Staining, Immunofluorescence, Expressing