Alexa Fluor® 488 Anti-Fibronectin antibody [F1]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 488 Anti-Fibronectin antibody [F1] (AB198933)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Alexa Fluor® 488 Anti-Fibronectin antibody [F1] (AB198933)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Alexa Fluor® 488 Anti-Fibronectin antibody [F1] (AB198933)

Immunofluorescence staining of Fibronectin 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.