IHC image of N cadherin staining in a section of formalin-fixed paraffin-embedded normal human heart performed on a Leica BOND^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab19348, 1 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

The inset secondary-only control image is taken from an identical assay without primary antibody.

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

ab19348 staining N-Cadherin in SH-SY5Y (Human neuroblastoma cell line from bone marrow) cells.

The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Tween for 5 mins 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 ab19348 at 5 μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

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

Immunofluorescence analysis of ARPE-19 (human retinal pigment epithelial) cells, staining N Cadherin (green) with ab19348, at 1/20 dilution.

ARPE-19 monolayer cultures were fixed in paraformaldehyde, permeabilized with 0.2% Triton X-100 for 15 min and blocked with 2% BSA for 30 min. Samples were incubated with primary antibody for 16 hours at 4°C before incubation with an Alexa Fluor^® 488-conjugated donkey anti-mouse secondary IgG for 60 min.