IHC image of CD15 staining in Human tonsil formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6). The section was incubated with ab17080, 1µg/ml, for 15 mins at room temperature.  A Goat polyclonal Secondary Antibody to Mouse IgM secondary antibody (ab97230) was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Human whole blood stained with ab17080 (right) or mouse IgM isotype control ab91545 (left). Red blood cells of 200ul human whole blood were lysed, then cells were incubated for 30 min on ice in 1x PBS containing 10ug/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab17080 ) or IgM isotype control (ab91545) (100ul at 1ug/ml) for 30 min on ice.

The secondary antibody Goat Anti-Mouse IgM mu chain (Alexa Fluor® 488) (ab150121) was used at 1/2000 dilution for 30 min at 4° C. Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were collected with the forward and side light-scatter characteristics of viable cells.

IHC image of CD15 staining in a section of frozen normal human spleen*. The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M 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 ab17080 at 1µg/ml. The section was then incubated with ab150117 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), 1/1000)) (shown in green) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. The secondary-only control insert image is taken from an identical assay without primary antibody. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.