Anti-CD68 antibody [FA-11] (ab53444)

Dual fluorescence combining IL-6 with markers for macrophages (CD68).

O, P, and Q, dual labeling of IL-6 (red) and marker of macrophage (green) in db/db mouse heart tissues. Arrows in Q show the specific CD68 staining with absence of IL-6 staining. (R and S) Negative control: arrows show the absence of staining in vessels with control IgG and without primary antibodies. (T) Staining of nuclei with DAPI (blue) in heart tissues of the db/db mice.

To identify and localize IL-6 protein in coronary arterioles, transverse sections of the mouse heart were stained using markers of endothelial cells, vascular smooth muscle cells, and macrophages. Freshly isolated hearts were embedded and frozen in OCT and sectioned at 5 μm. Slides were incubated with blocking solution (10% donkey serum in PBS) and permeabilized (0.1% Triton X-100 in PBS). Primary antibodies to IL-6 (goat polyclonal 15 micro g/ml, AF-406-NA; R&D) or macrophage marker CD68 (rat monoclonal, 1:1000, ab53444; Abcam) were used for sequential double immunofluorescence staining. Secondary antibodies were conjugated with the fluorophores FITC or Texas red. Sections were mounted in an anti-fading agent (Slowfade gold with DAPI; Invitrogen), and then the slides were observed and analyzed with a fluorescence microscope (IX81; Olympus) with a x40 objective (0.90 numerical aperture). For negative controls, primary antibodies were replaced with goat polyclonal IgG (Abcam), rabbit polyclonal IgG (GeneTex), and rat monoclonal IgG (Abcam) isotype controls at the same concentration. The specificity of the primary antibody was confirmed as the absence of immunofluorescence staining signals in the IL-6^-/- mice.

Diabetic mice (db/db).

Formaldehyde-fixed, frozen mouse lung tissue sections stained for CD68 using ab53444 at a 1/250 dilution in immunohistochemical analysis. Tissue sections were blocked using 1% BSA as a blocking agent for 10 minutes at 21°C. Primary antibody was incubated for 2 hours at 21°C. Secondary antibody was a biotin-conjugated goat anti-rat IgG at 1/250 dilution.

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IHC image of CD68 staining in mouse lung frozen tissue section. The section was incubated with ab53444, 0.1µg/ml, overnight at 4C. A goat anti-Rat biotinylated secondary antibody was used to detect the primary, and visualized using an ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

ab53444 stained RAW246.7 cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53444 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

ab53444 staining mouse heart tissue by Immunohistochemistry (IHC-frozen sections). Tissue underwent fixation in paraformaldehyde, no permeabilization and blocked in 1% serum for 10 minutes at 20°C. The primary antibody used undiluted and incubated with sample for 16 hours at 20°C. A Biotin conjugated goat polyclonal to rat Ig, diluted 1/100 was used as the secondary.  

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