Journal: Journal of the American Heart Association

Article Title: Successful Use of mRNA‐Nucleofection for Overexpression of Interleukin‐10 in Murine Monocytes/Macrophages for Anti‐inflammatory Therapy in a Murine Model of Autoimmune Myocarditis

doi: 10.1161/jaha.112.003293

Figure Lengend Snippet: Figure 6. Immunohistochemical staining. By trend, more IL-10, less macrophages (CD68), less infiltrating cells (hematoxylin -eosin), and less fibrosis were detected in IL-10–treated mice (n=4, respectively). MOCK indicates mock-transfected cells.

Article Snippet: A polyclonal rat anti-mouse CD68 antibody (dilution 1:50; clone FA-11, Acris Antibody GmbH, Herford, Germany) and a polyclonal goat anti-mouse IL-10 M-18 antibody (dilution 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) were used as a primary antibody.

Techniques: Immunohistochemical staining, Staining, Transfection

Journal: Journal of the American Heart Association

Article Title: Successful Use of mRNA‐Nucleofection for Overexpression of Interleukin‐10 in Murine Monocytes/Macrophages for Anti‐inflammatory Therapy in a Murine Model of Autoimmune Myocarditis

doi: 10.1161/jaha.112.003293

Figure Lengend Snippet: Figure 7. Colocalization of RFP with macrophages (CD68). a and b, In sequential sections, immunohistochemical staining for CD68 (brown) identifies RFP-positive cells (red) as macrophages (left). Previously these cells have been injected intravenously. 4′,6- Diamidino-2-phenylindole (DAPI) staining (blue) indicates the cell nuclei. Different magnifications are shown. Not all macrophages (brown) colocalize with the red cells as RFP is specific for intravenously injected donor macrophages. Consequently, macro- phages (brown) showing no RFP colocalization represent host macrophages. b, Right: Sections of the spleen served as a positive control for macrophage staining. Omitting the secondary antibody represented the negative control. RFP indicates red fluorescent protein.

Article Snippet: A polyclonal rat anti-mouse CD68 antibody (dilution 1:50; clone FA-11, Acris Antibody GmbH, Herford, Germany) and a polyclonal goat anti-mouse IL-10 M-18 antibody (dilution 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) were used as a primary antibody.

Techniques: Immunohistochemical staining, Staining, Injection, Positive Control, Negative Control

Journal: Basic Research in Cardiology

Article Title: Toll-like receptor-3 contributes to the development of aortic valve stenosis

doi: 10.1007/s00395-023-00980-9

Figure Lengend Snippet: AS development is impaired in TLR3-deficient mice: AS was induced in WT and TLR3 −/− mice via mechanical wire injury ( A ). Trans-aortic valve blood flow peak velocity ( B ) and mean gradient ( C ) did not increase in the same degree in TLR3 −/− animals as in wildtype mice after surgery. Left ventricular function remained unchanged in both groups ( D ) ( n = 19–20, *** p < 0.001, ** p < 0.01, * p < 0.05, mixed-effect model with Bonferroni multiple comparisons test, mean ± SEM). Plasma concentrations of pro- and anti-inflammatory cytokines ( E ). Histologic analysis of the aortic valve ( F ): H.E.-staining with quantitative analysis of the valve cusp area ( G ), CD68 staining: intra-valvular monocyte infiltration ( H ). FACS analysis of monocytes in peripheral blood of polyIC-treated mice (experimental design in Fig. A) and TLR3 −/− mice vs. control ( I ) (* p < 0.05, two-sided T tests, mean ± SEM). Quantification of valvular fibrosis ( J ) and calcification ( K ). ( n = 14–15, ** p < 0.01, two-sided T tests, mean ± SEM)

Article Snippet: The primary antibody was diluted 1:100 (anti-CD68 rat IgG2a, Acris Antibodies, Germany) and incubated at 4 °C overnight.

Techniques: Staining, Control

Journal: Basic Research in Cardiology

Article Title: Toll-like receptor-3 contributes to the development of aortic valve stenosis

doi: 10.1007/s00395-023-00980-9

Figure Lengend Snippet: Pharmacological TLR3 inhibition reduces AS formation in mice: Serum concentration of Regulated on activation, normal T cell expressed and secreted (RANTES) in mice 6 h after 100 µg polyIC injection and concomitant dose escalation C4a treatment ( A ) ( n = 2). RANTES serum concentration after C4a treatment and injection of increasing polyIC dose ( B ) (*** p < 0.001, * p < 0.05, two-way ANOVA with Tukey multiple comparisons test, mean ± SEM, n = 3). C4a or vehicle was injected in WT mice every second day after AS induction ( n = 20) ( C ). C4a inhibited increase in blood flow peak velocity ( D ) and mean gradient ( E ) compared to vehicle-treated mice. Left ventricular ejection fraction remained unchanged ( F ) (**** p < 0.0001, *** p < 0.001, mixed-effect model with Bonferroni multiple comparisons test, mean ± SEM). Aortic valve cusp area ( G ), infiltration of CD68 positive cells ( H ) and fibrosis ( J ) was significantly increased in C4a treated mice. Calcification was similar in both groups ( H ) ( n = 12–16, *** p < 0.001, ** p < 0.01, * p < 0.05, two-sided T tests, mean

Article Snippet: The primary antibody was diluted 1:100 (anti-CD68 rat IgG2a, Acris Antibodies, Germany) and incubated at 4 °C overnight.

Techniques: Inhibition, Concentration Assay, Activation Assay, Injection

Journal: Molecular Vision

Article Title: P2X 7 receptor activation may be involved in neuronal loss in the retinal ganglion cell layer after acute elevation of intraocular pressure in rats

doi:

Figure Lengend Snippet: Representative photomicrographs of double immunostaining of the P2X 7 receptor and CD68 in the normal retina and in the retina on days 1, 2, 3, and 7 after intraocular pressure elevation. P2X 7 -positive cells were also stained with anti-CD68 in the ganglion cell layer (GCL). Bar=100 µm.

Article Snippet: For this, mouse Alexa Fluor 488–labeled anti-TUJ1 monoclonal antibody (A488–435L, Covance Research Products, Princeton, NJ) and mouse anti-CD68 (MAB1435, Merck Millipore, Billerica, MA) were used.

Techniques: Double Immunostaining, Staining

Journal: Molecular Vision

Article Title: P2X 7 receptor activation may be involved in neuronal loss in the retinal ganglion cell layer after acute elevation of intraocular pressure in rats

doi:

Figure Lengend Snippet: Representative photomicrographs of double immunostaining of CD68 and TNF-α or interleukin-1β (IL-1 β) in the normal retina and the retina on day 2, with and without treatment of oxidized adenosine triphosphate (OxATP) at 30 µM just after intraocular pressure (IOP) elevation. A: TNF- α, B: IL-1 β. Immunoreactivities of tumor necrosis factor-α (TNF-α) and IL-1β were upregulated in the ganglion cell layer (GCL) and inner plexiform layer (IPL) cells on day 2 after IOP elevation; they were subsequently suppressed by treatment with OxATP. Bar=100 µm.

Article Snippet: For this, mouse Alexa Fluor 488–labeled anti-TUJ1 monoclonal antibody (A488–435L, Covance Research Products, Princeton, NJ) and mouse anti-CD68 (MAB1435, Merck Millipore, Billerica, MA) were used.

Techniques: Double Immunostaining