Immunofluorescence staining of PD1 staining in a section of formalin-fixed paraffin-embedded human normal 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°C 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 ab198933 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.

ab198933 staining Fibronectin in A431 (Human epidermoid carcinoma cell line) cells. The cells were fixed with 4% formaldehyde (10 minutes), 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 1 hour. The cells were then incubated overnight at +4°C with ab198933 at a 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 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).

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with ab198933 (red line). The cells were fixed with 4% formaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab198933, 1/50 dilution) for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG [EPR25A] Alexa Fluor^® 488 (ab199091) used at the same concentration and conditions as the primary antibody. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in A431 cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.