Journal: Nature Communications

Article Title: Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

doi: 10.1038/s41467-022-28777-9

Figure Lengend Snippet: A Experimental flow chart. B Representative immunostaining images of the transfection of GFP (green) reporter LV into astrocytes (GFAP, red) with enforced cytosolic LCN2 (blue) expression in the CC. White arrows indicated astrocytes transfected with LV. Scale bar, 20 μm. C , D Representative images and statistical analysis of C3d (blue) co-stained with GFAP (red) in mice receiving LV-NC or LV- Lcn2 (Δ2–20) on the 3rd and 7th day after dMCAO ( n = 5 mice; mean ± S.D.; * P < 0.05, *** P < 0.001 vs. LV-NC 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. E – G Representative confocal, 3D images and quantifications of Galectin3 + (blue) phagocytic astrocytes (GFAP + , red) internalizing myelin debris (dMBP + , green). ( n = 75 cells from five animals in each group for quantification of debris in a single astrocyte, n = 5 mice for others; mean ± S.D.; ** P < 0.01, *** P < 0.001 vs. LV-NC 3d; # P < 0.05, ## P < 0.01 vs. LV- Lcn2 (Δ2–20) 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. H , I Representative MBP, MAG, NF200 staining and quantifications from Lcn2 −/− dMCAO mice receiving LV ( n = 5 mice; mean ± S.D.; ** P < 0.01, ***P < 0.001 vs. LV-NC 3d; two-way ANOVA, Tukey post hoc test). Scale bar, 20 μm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan.

Techniques: Immunostaining, Transfection, Expressing, Staining

Journal: Nature Communications

Article Title: Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice

doi: 10.1038/s41467-022-28777-9

Figure Lengend Snippet: A Experimental flow chart in vivo. B Representative fluorescent images of the transfection of GFP (green) reporter LV in astrocytes (GFAP, blue) with reduced LRP1 (red) in the CC of WT mice. White arrows indicated astrocytes transfected with lentiviruses. Scale bar, 20 μm. C , D Immunoblotting analyses and quantifications for LCN2, LRP1, pLRP1, p38 and pp38 in vivo ( n = 5 mice; mean ± S.D.; ** P < 0.01, *** P < 0.001 vs. LC-NC; paired t -test). Protein samples derived from the same experiment and gels/blots were processed in parallel. E , F Representative images and quantifications showing the expression of LCN2 (red) and C3d (red) in astrocytes (GFAP, green) after dMCAO ( n = 5 mice; mean ± S.D.; paired t -test). Scale bar, 20 μm. G – J Representative confocal, 3D reconstruction images and quantifications of phagocytic astrocytes (Galectin3 + , blue; GFAP + , red) engulfing dMBP + (green) debris in mice receiving LC-NC or LV- Lrp1 -RNAi. ( n = 75 cells from five animals in each group for quantification of debris in single astrocyte, n = 5 mice for others; mean ± S.D.; * P < 0.05, *** P < 0.001 vs. LC-NC; paired t -test). Scale bar, 20 μm. K Experimental flow chart in vitro. L Representative immunocytochemistry images of GFAP (green), LRP1 (red), LCN2 (blue) and DAPI (cyan) after lentiviruses transfection. Scale bar, 20 μm. M – P Representative confocal images, ELISA and flow cytometry analyses showing the differences of astrocytic phagocytosis between the LC-NC and LV- Lrp1 -RNAi groups in vitro ( n = 5 independent primary cell cultures; mean ± S.D.; * P < 0.05 vs. LC - NC; paired t -test). Scale bar, 20 μm. In the box plots ( N , P ), the middle bar represents the median, the box represents the interquartile range and whiskers indicate the maximum and minimum values. Dots are all the data points. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan.

Techniques: In Vivo, Transfection, Western Blot, Derivative Assay, Expressing, In Vitro, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Flow Cytometry