Recombinant Alexa Fluor® 488 Anti-CD68 antibody [KP1] (ab222914)

ab222914 staining CD68 in THP-1 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab222914 at 1/100 dilution (shown in Green) and ab190573, Rabbit monoclonal to alpha Tubulin (Alexa Fluor® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow cytometry overlay histogram showing left THP1 positive cells and right negative Jurkat cells stained with ab222914 (red line). The cells were fixed with 4% formaldehyde (10 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 (ab222914) (1x 10⁶ in 100 µL at 0.04 µg/mL (1/12500)) for 30 min at 22°C.

Isotype control antibody (black line) was mouse IgG1κ Alexa Fluor^® 488 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 THP1 cells fixed with 80% methanol (5 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

IHC image of CD68 staining in a section of formalin-fixed paraffin-embedded normal human tonsil*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110^oC for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab222914 at 1/100 dilution (shown in green) and counterstained using ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.