重组Alexa Fluor® 488荧光Anti-CD68抗体[KP1] (ab222914)

Immunofluorescence analysis of human bladder transitional cell carcinoma labelled with ab222914. Image was acquired using Cell DIVE Multiplexed Imaging Solution (Leica Microsystems).

Formalin fixed paraffin embedded (FFPE) sections were incubated with ab222914 at 1/100 dilution, for 1h at room temperature. Nuclear DNA was labelled with DAPI (Red). Deparaffinization, antigen retrieval, biomarker labeling and imaging was performed following Cell DIVE recommended standard protocol. Cell DIVE automatically processed the image to perform autofluorescence removal, distortion correction, blank glass subtraction, flat-field correction, and stitching. 

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.