Journal: Translational psychiatry

Article Title: Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

doi: 10.1038/tp.2017.80

Figure Lengend Snippet: Figure 4. Signs of classical pro-inflammatory activation in Poly(I:C) hippocampal microglia. (a) Representative image of a sagittal slice (from a control animal) used for the analysis of the binding capacity with the TSPO-specific radioligand [18F]GE180. (b) Shows a representative image of a Nissl-stained sagittal slice (control animal) used to localize specific brain regions. (c) Shows that Poly(I:C) mice exhibit an increased binding potential to the TSPO in the hippocampus (n = 5 mice) with respect to controls (n = 6 mice). On the other hand, slices from Poly(I:C) mice treated with minocycline display a normalized binding potential in the latter region (n = 6 mice), significantly lower than untreated Poly(I:C) animals and comparable to controls. (d) Representative pictures illustrating increased Iba1 immunoreactivity in the proximity of the DG of Poly(I:C) mice as compared with controls and minocycline-treated mice. Pictures were taken at a 63-times magnification. (e) Iba1 immunoreactivity was increased in the dentate gyrus of the hippocampus (DG) of Poly(I:C) mice (n = 7) as compared with controls (n = 5). Poly (I:C) animals treated with minocycline (n = 4) displayed a normal Iba1 immunoreactivity. (f–h) Enzyme-linked immunosorbent assay (ELISA) measurement in whole hippocampal homogenates of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Only IL-6 was found to be significantly increased (h), while the levels of the other cytokines remained unchanged (f and g; Controls, n = 5; Poly(I:C), n = 5; Poly(I:C) treated with minocycline, n = 4). Error bars represent s.e.m. in all the panels. The data from the radioligand-binding assay were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The data from the immunoreactivity and ELISA were analyzed by one-way ANOVA followed by Newman–Keuls post hoc test. *Po0.05, **Po0.001. Cb, cerebellum; Cc, corpus callosum; ctrl., control animals; Ctx, cortex; Hip, hippocampus; Ob, olfactory bulbs; Poly, Poly(I:C) animals; Poly/Mino, Poly(I:C) animals treated with minocycline; Th, thalamus; TSPO, translocator protein.

Article Snippet: Complement component 4a (C4a) levels in hippocampal lysates were measured using the mouse C4a enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology, Wuhan, China), according to the manufacturer’s manual.

Techniques: Activation Assay, Control, Binding Assay, Staining, Enzyme-linked Immunosorbent Assay, Radio Ligand Binding Assay

Journal: Translational psychiatry

Article Title: Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

doi: 10.1038/tp.2017.80

Figure Lengend Snippet: Figure 5. Changes in the complement system in the hippocampus of Poly(I:C) mice. (a) Enzyme-linked immunosorbent assay (ELISA) measurement of the complement component 4a (C4a) in whole hippocampal homogenates from Poly(I:C) (n = 5), control (n = 5) and minocycline-treated Poly(I:C) animals (n = 4). No significant difference in C4a levels was detected between the groups. (b) Immunoreactivity for CD18 on microglia measured through the dentate gyrus (DG) of the hippocampus (DG) of Poly(I:C) (n = 7), controls (n = 5) and minocycline- treated Poly(I:C) mice (n = 4). CD18 immunoreactivity in Iba1-positive cells was significantly increased in the DG of Poly(I:C) animals as compared with controls and minocycline-treated Poly(I:C) mice. (c) Representative pictures showing the CD18 signal co-localizing with Iba1- positive cells (microglia) and showing the increased CD18 immunoreactivity in the proximity of the DG in Poly(I:C) animals as compared with controls and minocycline-treated Poly(I:C) mice. Error bars represent s.e.m. in all the panels. The data were analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls post hoc test; *Po0.05; ctrl, control animals; NS, not significant; Poly, Poly(I:C) animals; Poly/ Mino, Poly(I:C) animals treated with minocycline.

Article Snippet: Complement component 4a (C4a) levels in hippocampal lysates were measured using the mouse C4a enzyme-linked immunosorbent assay (ELISA) kit (Elabscience Biotechnology, Wuhan, China), according to the manufacturer’s manual.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Complement deposition and histopathologic changes in retinas of aged Tspan12 ECKO mice. A , Complement factor C4 deposition around retinal blood vessels, inner nuclear layer (INL), and outer plexiform layer (OPL). White arrows point to some of the small cystoid lesions decorated with complement. B , H&E staining of a control retina and a relatively strongly affected area of a mutant retina reveals cystoid lesions consistent with edema. C , Increased F4/80 staining on microglia of mutant retinas. D through F , Extravastion of IgG, transferrin, and albumin in Tspan12 ECKO retinas. G , COL4 (collagen IV) staining in retinal blood vessels of 12-mo-old ECKO mice is increased, staining in the inner limiting membrane (white arrowheads) serves as internal staining control.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Control, Mutagenesis, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Endothelial Cell–Specific Inactivation of TSPAN12 (Tetraspanin 12) Reveals Pathological Consequences of Barrier Defects in an Otherwise Intact Vasculature

doi: 10.1161/ATVBAHA.118.311689

Figure Lengend Snippet: Colocalization of complement factor C4 with several retinal cell types. A , Twelve-mo-old Tspan12 ECKO retinal cross-sections stained with anti-C4 and IB4. Split and merged channels are shown. Arrows indicate blood vessels decorated with C4. B , Colocalization of extravasated IgG and C4 on a subset of retinal cells and on the borders of cystoid lesions in the mutant tissue. C , Rod-bipolar cell spacing seems increased in the mutant tissue. The section shows also 1 or 2 C4-decorated Müller glia cells, which were recognized by their extensions towards the outer limiting membrane (arrow). D , Disorganized Müller cells in the mutant tissue. Sox9 marks nuclei of Müller glia. Arrow highlights a C4-decorated cell which was recognized as amacrine cell based on cell body location and cell shape. E , Partial colocalization of C4 with microglia marker IBA-1. PKC indicates protein kinase C; and Sox9, SRY-box 9.

Article Snippet: The following primary antibodies and lectins were used for this study: GS Isolectin B4 Alexa-488 conjugate 1:100 (Invitrogen, I21411), rat PECAM (platelet endothelial cell adhesion molecule) 1:50 (BD, 550274), rat PLVAP 1:50 (BD, 550563), rabbit desmin 1:200 (Cell Signaling, D93F5), rat vascular endothelial (VE)-cadherin 1:100 (BD, clone 11D4), rat complement component C4 1:200 (Cedarlane, CL7504AP), goat transferrin 1:200 (R&D, AF3987), rabbit COL4 (collagen IV) 1:200 (Abcam, 6586), goat albumin 1:200 (Bethyl, A90-234A), goat IBA1 1:200 (Novus, NB100-1028), rat F4/80 1:200 (AbD Serotec, MCA497R), rabbit PKC (protein kinase C) α 1:200 (Santa Cruz, C-20), and rabbit Sox9 1:200 (Millipore, AB5535).

Techniques: Staining, Mutagenesis, Membrane, Marker

Journal: bioRxiv

Article Title: Single-cell atlas of pig-to-monkey kidney xenotransplantation reveals macrophage chimerism and an IFN-ε orchestrated graft protective immune niche

doi: 10.64898/2026.03.21.713416

Figure Lengend Snippet: (A) Haematoxylin and eosin-based evaluation for xenotransplantation control #1 (sections a1-a3) and #2 (sections c1-c3), xenograft #1 (sections b1-b3) and #2 (d1-d3). Fields representative of glomeruli (row 1), peritubular capillaries (row 2), and tubulointerstitial compartments (row 3) are shown. Black arrows show non-infiltrated peritubular capillaries (sections a2 and c2), and infiltrated peritubular capillaries (section b2 and d2). (B) Immunohistochemistry-based evaluation for xenotransplantation control #1 (sections a1-a3) and #2 (sections c1-c3), xenograft #1 (sections b1-b3) and #2 (d1-d3). Fields representative of IgM (row 1), IgG (row 2) and C4d (row 3) stainings are shown. (C) Immunofluorescence staining of infiltrated macrophages with signature proteins. Scale bars, 100 µm.

Article Snippet: The primary antibody panel including: anti-IgM (11016-1-AP, 1:100, Proteintech), anti-IgG (ab109489, 1:500, Abcam), C4d (22233-1-AP, 1:200, Proteintech), anti-CD31 (ab28364, 1:50, Abcam), anti-vWF (ab6994, 1:200, Abcam), anti-CD3 (GB12014, 1:1000, Servicebio) and anti-CD15 (YT0726, 1:200, Immunoway) for IHC; anti-CD68 (bs-0649R, 1:200, Bioss), anti-SPP1 (83341-1-RR, 1:200, Proteintech), anti-VEGFA (66828-1-Ig, 1:200, Proteintech), anti-PD-L1 (ab205921, 1:200, Abcam), anti-IDO1 (ab211017, 1:1000, Abcam), anti-ARG1 (66129-1-Ig, 1:200, Proteintech), anti-FCGR2B (NB100-65338, 1:100, Novus Biologicals), anti-CD8a (66868-1-Ig, 1:400, Proteintech), and anti-KLRD1 (84466-5-RR, 1:200, Proteintech) for multiplexed IF.

Techniques: Control, Immunohistochemistry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1101/598342

Figure Lengend Snippet: (A) Diagram depicting IUE surgery performed in E16 dams. (B) Representative 20X confocal image of IUE with GFP targeted to L2/3 mPFC. Yellow asterisk: L2/3 GFP+ neurons. Left panel scale bar = 250 μm. Right panel scale bar = 75 μm. (C) Representative 60X confocal images of in situ hybridization from the same coronal section showing CaMKIIα+ neurons that are transfected with GFP and mC4 (C4B) (white arrowhead) and untransfected neighbors expressing mC4 (pink arrowhead) in P21 mice. Scale bar = 15 μm. (D) IUE reliably increases C4B transcript level in transfected cells. Percent of soma area positive for C4 transcript in transfected and untransfected neurons. N = 100 neurons (3 mice) per condition. t-test. **** p < 0.0001. Mean and SEM. (E) Transcript levels of GFP and mC4 positively correlate in transfected cells. Black line: linear fit. Gray lines: 95% confidence intervals. Black dotted line: average endogenous C4 expression in CaMKIIα+ mPFC L2/3 neurons. N = 100 transfected neurons (3 mice). Pearson’s R correlation and linear regression. R 2 = 0.28. **** p < 0.0001.

Article Snippet: DNA sequences containing mouse C4B (NM_009780.2, synthesized by Genescript) and human C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: In Situ Hybridization, Transfection, Expressing

Journal: bioRxiv

Article Title: Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction

doi: 10.1101/598342

Figure Lengend Snippet: (A) Developmental time course reveals significant decrease of spine density in neurons overexpressing C4 at P21-23. ** p < 0.01, **** p < 0.0001. (B) Representative 40X confocal images of P21-23 apical dendritic tufts. Scale bar = 5 μm. (C) Representative 40X confocal images of P21-23 apical dendritic spine types. Yellow asterisk: large mushroom spine (TIB (a.u.) > 75%). Green arrowhead: thin spine/filopodia (TIB (a.u.) < 25%). Scale bar = 3 μm. (D) Spine density sorted by spine types (spine/μm) reveals a specific reduction of medium-sized and thin/filopodia spine types in the mC4 condition. ** p < 0.01. (E) Representative 40X confocal images of P21-23 basal dendritic spines. Scale bar = 5 μm. (F) Analysis of basilar spine density (spine/μm) reveals no difference across groups. (A-F) N = 10-12 dendrites (from 4 mice) for each condition. Two-way ANOVA and Tukey’s test. Control: blue, mC4: red, hC4: purple. Mean and SEM.

Article Snippet: DNA sequences containing mouse C4B (NM_009780.2, synthesized by Genescript) and human C4A (RC235329, Origene) were subcloned (InFusion Kit, Clonetech) into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively.

Techniques: