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

IHC image of CD68 staining in a section of frozen mouse lung performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab53444, 0.1ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

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).

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.  

See Abreview

See Abreview

See Abreview

Flow cytometry overlay histogram showing RAW 264.7 cells stained with ab53444 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab53444) (1x10⁶ in 100µl at 0.2µg/ml) for 30 min at 22°C. The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor 488®, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody (black line) was Rat IgG2a k (ab18450) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in RAW 264.7 cells fixed with 4% formaldehyde (10 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.