Journal: bioRxiv

Article Title: The cGAS-STING pathway is dispensable in a mouse model of LMNA -cardiomyopathy despite nuclear envelope rupture

doi: 10.1101/2023.08.28.555134

Figure Lengend Snippet: A) Tamoxifen treatment (Tam) deletes Lmna in cardiomyocytes in mice at 6-8 weeks of age. B) Top: Lamin A/C immunoblot for cardiomyocytes isolated from two wild-type and three Lmna CKO mice. Bottom: Quantification of immunoblot signals. C) Lamin A/C immunohistochemistry (brown) with hematoxylin counterstaining (blue) in mouse heart tissues (60×). Scale bar: 20 μm. D) Hematoxylin/eosin staining of whole hearts. Scale bar: 1 mm. E) Left ventricular ejection fraction (EF) measured by echocardiography. Dot, EF of individual mice. Boxplot, interquartile range. F) Kaplan-Meier survival plot. WT, n=48. Lmna CKO , n=42. G) Volcano plot contrasting gene expression in Lmna CKO versus WT left ventricles, determined by RNA-seq. Upregulated (up) or downregulated (down) genes at P < 0.05 with Log 2 fold change > 1. H) The top 10 Gene Ontology terms overrepresented among differentially expressed genes. I) Normalized gene expressed levels (TPM) from RNA-seq data for the cGAS-STING pathway-related genes. Dot, individual mouse TPM. Bar, mean of biological replicates (WT, n=7. Lmna CKO , n=5). J) Immunofluorescence of heart tissue sections for CD45 (pan-leukocyte, red) and CD68 (macrophage, green). Scale bar: 20 μm. Quantification, . K) Masson’s trichrome staining of heart sections. Arrow, collagen deposition. Scale bar: 20 μm. L) Timeline of events.

Article Snippet: Antigen-retrieved sections were incubated overnight at 4 °C with goat anti-CD45 antibody (R&D Systems, AF114-SP) and rabbit anti-CD68 antibody (Cell Signaling, 97778), or rabbit anti-γ-H2A.X antibody (Cell Signaling, 9718) and goat anti-desmin antibody (Invitrogen, PA5-19063), followed by Alexa fluorophore-conjugated secondary antibodies for 1 hour at 37 °C.

Techniques: Western Blot, Isolation, Immunohistochemistry, Staining, Gene Expression, RNA Sequencing, Immunofluorescence

Journal: bioRxiv

Article Title: The cGAS-STING pathway is dispensable in a mouse model of LMNA -cardiomyopathy despite nuclear envelope rupture

doi: 10.1101/2023.08.28.555134

Figure Lengend Snippet: A) Left: Lmna LoxP allele and the expected recombined allele by Cre expression. Arrow, PCR primer location. Right: PCR detection of the recombined (deleted) allele in Lmna CKO cardiomyocytes. Recombination (%), 153-bp band (Recombined) over 598-bp band (Floxed) intensities. B) Whole immunoblot for Lamin A/C and GAPDH, and Coomassie staining of original gel, in isolated cardiomyocytes. Part is shown in . C) M-mode echocardiography of left ventricular (LV) geometry. P, Wilcoxon test. D) Quantification of CD45 and CD68-positive cells in heart sections. Representative image in .

Article Snippet: Antigen-retrieved sections were incubated overnight at 4 °C with goat anti-CD45 antibody (R&D Systems, AF114-SP) and rabbit anti-CD68 antibody (Cell Signaling, 97778), or rabbit anti-γ-H2A.X antibody (Cell Signaling, 9718) and goat anti-desmin antibody (Invitrogen, PA5-19063), followed by Alexa fluorophore-conjugated secondary antibodies for 1 hour at 37 °C.

Techniques: Expressing, Western Blot, Staining, Isolation

Journal: Developmental cell

Article Title: Mouse placenta fetal macrophages arise from endothelial cells outside the placenta.

doi: 10.1016/j.devcel.2022.11.003

Figure Lengend Snippet: Figure 1. Mouse placental fMacs are located in the perivascular space and expand through local proliferation (A) Schematics of the mouse mature placenta (left) and labyrinth (right) are shown. Thin pink lines on the left indicate fetal blood vessels. Jz, junctional zone; Lab, labyrinth; CA, chorioallantoic region; SynT-I, type I syncytiotrophoblasts; SynT-II, type II syncytiotrophoblasts; S-TGC, sinusoidal trophoblast giant cells; mHC, maternal hematopoietic cells; EC, endothelial cell; fMac, fetal macrophage. (B) Immunofluorescence staining for F4/80 (green) and CD45 (red) on wild-type E13.5 placenta sections (n = 3). White arrows indicate F4/80+CD45+ placental fMacs. Red arrows indicate maternal hematopoietic cells (mHCs). (C) Immunofluorescence staining for F4/80 (green) and CD45 (red) on wild-type E10.5, E11.5, E13.5, and E17.5 placenta sections (n = 3). Left two panels show the chorioallantoic region, whereas the right two panels show staining in the labyrinth. White arrows indicate F4/80+CD45+ macrophages in E11.5 labyrinth. Dotted line indicates boundary between labyrinth and CA. (D) Quantification of F4/80+CD45+ placental fetal macrophages detected by immunostaining of placenta tissue sections. Each dot represents the number of detected cells in sections from middle part of one placenta. (E) Immunofluorescence staining for F4/80 (blue) and MCT4 (red) and MCT1 (green) on E13.5 wild-type placenta (n = 2). White arrows indicate fetal macrophages.

Article Snippet: The following primary antibodies were used for immunostaining: rabbit anti-RFP (1:200, 600-401-379, Rockland), Goat anti-GFP (1:200, ab6673, Abcam), Rabbit anti-F4/80 (1:100, 70076, CST) (specific for paraffin section), Rat anti-F4/80 (1:200, ab6640, Abcam) (specific for frozen section), Rabbit anti-CD68 (1:200, 97778, CST), Goat anti-Endomucin (1:200, AF4666, R&D), Rat anti-Endomucin (1:100, sc-65495, Santa Cruz), Goat anti-CD45 (1:200, AF114, R&D), Chicken anti-MCT1 (1:200, AB1286-I, Sigma), Rabbit anti-MCT4 (1:200, AB3316P, Sigma) and Rabbit anti-Col1a1 (1:200, 72026, CST).

Techniques: Staining, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response

doi: 10.1186/s12974-016-0753-x

Figure Lengend Snippet: Fluorescence microscopy analysis of the cellular inflammatory response during the subacute phase of stroke. Coronal sections of the unlesioned ( a ) and lesioned ( b , c ) hemispheres of mice subjected to dMCAO, at 48 h after surgery. The sections were immunostained with anti-Iba-1 antibody ( red ). c Peri-infarct area of stroke; Infarct core (I) is outlined with white dotted line . Note the transformation of microglial phenotype from ramified to hypertrophic bushy and ameboid, as their position is closer to the infarct border. d – f 7 days after dMCAO. Triple immunostaining with the antibodies against GFAP ( blue ), Iba-1 ( green ), and NeuN ( red ). d Infarct core (I), glial scar, and peri-infarct area. The edge of the infarct is marked with white arrowheads . e , f Higher magnification images demonstrate the distribution of microglia ( green ), astrocytes ( blue ), and neurons ( red ) in the peri-infarct area and the immediate vicinity to glial scar. g – i 21 days after dMCAO. g The infarct core (I), glial scar, and peri-infarct area. The edge of the infarct is marked with white arrowheads . Higher magnification images demonstrate the distribution of microglia ( green ), astrocytes ( blue ), and neurons ( red ) in the peri-infarct area ( h ). Note the narrowing of the glial scar and its concentration mainly in the white matter area ( i ). Imaging was performed using Zeiss LSM510-META confocal microscope, using single-scan and tile-scan image acquisitions. Bars : a – c 20 μm; d , g 100 μm; e , h 10 μm; f , i 50 μm

Article Snippet: Other antibodies used were as follows: mouse monoclonal anti-STAT-3 (Cell Signaling Technology Cat# 9139, RRID:AB_331757); rabbit polyclonal anti-SOCS-1 (Cell Signaling Technology Cat# 3950S, RRID:AB_2192983); anti-SHIP-1 (Cell Signaling Technology Cat# 2728, RRID:AB_2126244); anti-C/EBP-β (Cell Signaling Technology Cat# 3087, RRID:AB_2078052); rabbit polyclonal anti-SOCS-6 (Santa Cruz Biotechnology Cat# sc-5608, RRID:AB_661195); rabbit polyclonal anti-Iba-1 (Wako Cat# 019-19741, RRID:AB_839504); rat anti-mouse CD68 (AbD Serotec Cat# MCA1957, RRID:AB_322219); goat polyclonal anti-CD206 (R and D Systems Cat# AF2535, RRID:AB_2063012); and anti-CD45 (R and D Systems Cat# AF114, RRID:AB_442146).

Techniques: Fluorescence, Microscopy, Transformation Assay, Triple Immunostaining, Concentration Assay, Imaging

Journal: Journal of Neuroinflammation

Article Title: In vivo inhibition of miR-155 significantly alters post-stroke inflammatory response

doi: 10.1186/s12974-016-0753-x

Figure Lengend Snippet: Analysis of M/M phenotypic markers at different time points after dMCAO. a Coronal sections of the lesioned hemispheres of the inhibitor and control mice brains, at seven ( two upper rows of panels ) and 14 days ( two lower rows ) after dMCAO. The sections were triple-immunostained with anti-Iba-1 ( red ), anti-CD45 ( blue ), and anti-CD68 ( green ) antibodies. Representative merged and single-channel images depict part of the infarct core (I, outlined with white dotted lines ) and peri-infarct area of stroke. High magnification images demonstrate distribution of Iba-1, CD45, and CD68 in the glial scar area ( right panels ). Imaging was performed using Zeiss LSM800 Airyscan confocal microscope, using single-scan and tile-scan image acquisitions. Bars : from left to right: 100 and 10 μm. b Quantification of the corrected fluorescence intensity for CD45 and CD68 immunofluorescence staining in the inhibitor ( black bars ) and control groups ( white bar ), at 7 and 14 days after dMCAO. N = 5–6 mice per group/per time point, three brain sections per mouse. Error bars : SEM; Student’s t test, * p < 0.05; ** p < 0.01. c Representative Western blots and quantification analysis ( graphs ) of CD206, CD45, CD68, and Iba-1 expression in lysates prepared from the lesioned cortical tissue of the animals from the inhibitor ( black bars ) and control ( white bars ) groups, at 7 and 14 days after dMCAO. Graphs : optical density of each band was normalized to β-actin (loading control). N = 5–6 mice per group/per time point. Error bars : SEM; Student’s t test, * p < 0.05

Article Snippet: Other antibodies used were as follows: mouse monoclonal anti-STAT-3 (Cell Signaling Technology Cat# 9139, RRID:AB_331757); rabbit polyclonal anti-SOCS-1 (Cell Signaling Technology Cat# 3950S, RRID:AB_2192983); anti-SHIP-1 (Cell Signaling Technology Cat# 2728, RRID:AB_2126244); anti-C/EBP-β (Cell Signaling Technology Cat# 3087, RRID:AB_2078052); rabbit polyclonal anti-SOCS-6 (Santa Cruz Biotechnology Cat# sc-5608, RRID:AB_661195); rabbit polyclonal anti-Iba-1 (Wako Cat# 019-19741, RRID:AB_839504); rat anti-mouse CD68 (AbD Serotec Cat# MCA1957, RRID:AB_322219); goat polyclonal anti-CD206 (R and D Systems Cat# AF2535, RRID:AB_2063012); and anti-CD45 (R and D Systems Cat# AF114, RRID:AB_442146).

Techniques: Imaging, Microscopy, Fluorescence, Immunofluorescence, Staining, Western Blot, Expressing