ab53444 staining CD68 in Raw264.7 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% n

normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab53444 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150165, Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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 staining CD68 in mouse spleen tissue by Immunohistochemistry (Frozen sections). Antibody was detected with HRP-conjugated Goat anti-Rat IgG, showing staining in the red pulp.

Staining of permeabilised mouse peritoneal macrophages with Rat anti mouse CD68 (ab53444).

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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ab53444 at 1/100 dilution staining CD68 in mouse spleen tissue by immunohistochemistry (frozen sections). Sections were acetone fixed prior to blocking in 8% milk for 40 minutes at 36°C and then incubated with ab53444 for 20 hours at 4°C. A biotin conjugated goat polyclonal to rat Ig, diluted 1/400, was used as the secondary antibody.

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ab53444 staining CD68 in murine heart tissue by Immunohistochemistry (Frozen sections).
Tissue was fixed in acetone, permeabilized using 0.3% Triton, blocked with 10% serum for 30 minutes at 20°C, then incubated with ab53444 at a 1/1000 dilution for 16 hours at 4°C. The secondary used was a FITC conjugated goat anti-rat polyclonal, used at a 1/1000 dilution.

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