goat anti cd45  (R&D Systems)

Journal: bioRxiv

Article Title: Lymphatic activation of ACKR3 signaling regulates lymphatic response after ischemic heart injury

doi: 10.1101/2024.12.04.626683

Figure Lengend Snippet: A) Venn diagram summarizing the transcriptomic differences in cardiac LECs isolated from male Ackr3 fl/fl and Ackr3 ΔLyve mice one week post LAD ligation. B) Differentially expressed genes in cardiac LECs upon LAD ligation that are specific to ACKR3-deficient mice. Positive fold change values represent upregulation in LAD ligated Ackr3 ΔLyve LECs. C) Comparison of gene expression levels of identified ACKR3-specific LAD-triggered genes between LAD ligated Ackr3 ΔLyve and Ackr3 fl/fl mice. Positive fold change values represent upregulation in LAD ligated Ackr3 ΔLyve LECs. D) Visualization of LYVE1-positive cardiac lymphatic vessels of male Ackr3 fl/fl and Ackr3 ΔLyve whole mount hearts 6- and 28-days post LAD ligation. Representative images of from 3-4 animals per group. Bars, 500 µm. E,F) Quantification of lymphatic vessel length (E) and branching index (number of branches divided by lymphatic vessel length) (F) per field of view. N=3-4 per group. Two-way ANOVA, Dunnett’s post-hoc test. G) LYVE1 (cyan) and CD45 (magenta) staining of the infarct zone and peri-infarct zones of male Ackr3 fl/fl and Ackr3 ΔLyve hearts 28-days post LAD ligation. Yellow arrows point at LYVE1-positive lymphatic structures in the injured tissues of the infarct and peri-infarct zone of hearts. White arrowheads point at epicardial lymphatic structures. Representative images from N=3-6 mice per group. Bars, 200 µm. H,I) Area-adjusted lymphatic vessel counts in the infarct zone (H) and percent area of the infarct zone occupied by lymphatic vessels (I) in male Ackr3 fl/fl and Ackr3 ΔLyve hearts 28-days post LAD ligation. N= 3-6 per group. Unpaired Welch’s unequal variances t-test.

Article Snippet: Immunofluorescent staining was performed using the following primary and secondary antibodies: rabbit anti-LYVE1 (1:200, Fitzgerald, 70R-LR005), goat anti-CD45 (1:50, Novus Biologicals, NB100-77417), goat anti-F4/80 (1:50, BD Pharminogen, 565409), donkey anti-rabbit Cy3 (1:200, Jackson ImmunoResearch, 711-165-152), donkey anti-goat Cy5 (1:200, Jackson ImmunoResearch, 705-175-147).

Techniques: Isolation, Ligation, Comparison, Expressing, Staining

Journal: bioRxiv

Article Title: Endothelial RIPK3 minimizes organotypic inflammation and vascular permeability in ischemia-reperfusion injury

doi: 10.1101/2024.11.25.625188

Figure Lengend Snippet: ( A ) Representative images of immortalized BMA3.1A7 macrophages labeled with calcein-AM fluorescent dye (green) and incubated on top of a monolayer of WT and Ripk3 -KO MS1 ECs that were previously treated with 20ng/mL TNFα for 24 hr. Cells were washed multiple times before imaging. Scale bar: 20 µm. ( B ) Following imaging (as in A), cells were lysed with Triton-X, and fluorescence was measured using a plate reader. Values were normalized to readings from wells in which BMA3.1A7 cells were incubated with untreated WT MS1-ECs (dotted line) (n=3/group). ( C ) CD45 + leukocyte accumulation was assessed by immunostaining in various tissues from Ripk3 WT and Ripk3 iECKO mice following 24 hr I/R injury. Scale bar: 100 µm. CD45 + cells were quantified from multiple confocal Z-stack images taken from each mouse tissue by normalizing CD45 + expression area to total cell nuclear area in small intestines ( D ), lungs ( E ), kidneys ( F ) and livers ( G ) of Ripk3 WT and Ripk3 iECKO mice. Individual data points are averages of 2-4 imaged and quantified sections per mouse (n=3 mice/group). P values were determined by unpaired t-tests (B, D, E, F and G ). * P <0.05 vs . Control. Summary data are the mean ± SE.

Article Snippet: Primary antibodies used for immunofluorescence were rabbit-anti-GFP (1:200, Novus Biological; #NB600-303), rat-anti-VE-cadherin (1:100, Abcam; #ab91064), goat-anti-CD45 (1:50, R&D Systems; #AF114), goat-anti-VCAM-1 (1:100, R&D Systems; #AF643), goat-anti-ICAM-1 (1:100, R&D Systems; #AF796), rabbit-anti-NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) p65 (1:700, Cell Signaling; #8242), goat-anti-CD31 (1:100, R&D Systems; #AF3628), rat-anti-CD31 (1:100, BD Pharmingen; #553370) and rabbit-anti-NRF2 (1:300, gift from Dr. Scott Plafker at OMRF) .

Techniques: Labeling, Incubation, Imaging, Fluorescence, Immunostaining, Expressing, Control

Journal: Cell reports

Article Title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

doi: 10.1016/j.celrep.2024.114284

Figure Lengend Snippet: (A) Tamoxifen (Tam) induces Lmna deletion in cardiomyocytes in adult mice. See for related analyses. (B) Lamin A/C immunoblot in isolated cardiomyocytes (top) with signal quantification (bottom). n = 2–3 mice/genotype. (C) Lamin A/C immunohistochemistry (brown) with hematoxylin counterstaining (blue) in mouse heart tissues. Scale bar: 20 μm. (D) Hematoxylin and eosin staining of hearts. Scale bar: 1 mm. (E) Left ventricular ejection fraction with interquartile range (box) measured by echocardiography. p value, Wilcoxon test. n = 5–10 mice/genotype. (F) Kaplan-Meier survival analysis. Wild type (WT) n = 48, Lmna CKO n = 42. p value, log-rank test. (G) Volcano plot comparing RNA-seq read counts in Lmna CKO versus WT hearts. Number, upregulated or downregulated gene count. n = 3–7 mice/genotype. (H) Top 10 Gene Ontology (GO) terms overrepresented among differentially expressed genes. p value is computed by Metascape. (I) Transcript abundance in hearts quantified by RNA-seq. TPM, normalized transcripts per million. Bar, mean. n = 5–7 mice/genotype. P, false discovery rate (FDR)-adjusted p value computed by a generalized linear model in DESeq2. (J) Immunofluorescence of heart tissue sections for CD45 (pan-leukocyte, red) and CD68 (macrophage, green). Scale bar: 20 μm. (K) Masson’s trichrome staining of heart sections. Arrow, collagen deposition. Scale bar: 20 mm. (L) Summary.

Article Snippet: Deparaffinized heart tissue sections, that had been fixed with 10% formalin, were subjected to heat-induced antigen retrieval in 10 mM Tris EDTA buffer (pH 9.0) for 5 min. 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), or rabbit anti-PCM1 antibody (Sigma, HPA023370), goat anti-TdTomato antibody (OriGene, AB8181), and mouse anti-Cox4 antibody (R&D Systems, MAB6980), followed by Alexa fluorophore-conjugated secondary antibodies for 1 h at 37°C.

Techniques: Western Blot, Isolation, Immunohistochemistry, Staining, RNA Sequencing Assay, Immunofluorescence

Journal: Cell reports

Article Title: Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

doi: 10.1016/j.celrep.2024.114284

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Deparaffinized heart tissue sections, that had been fixed with 10% formalin, were subjected to heat-induced antigen retrieval in 10 mM Tris EDTA buffer (pH 9.0) for 5 min. 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), or rabbit anti-PCM1 antibody (Sigma, HPA023370), goat anti-TdTomato antibody (OriGene, AB8181), and mouse anti-Cox4 antibody (R&D Systems, MAB6980), followed by Alexa fluorophore-conjugated secondary antibodies for 1 h at 37°C.

Techniques: Recombinant, Transfection, Plasmid Preparation, Electron Microscopy, Modification, TUNEL Assay, In Situ, Generated, Clone Assay, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy