Western Blot analysis of Syndecan-2 using S9111-40B. Lane 1: K562 Lane 2: HT-29 Lane 3: Hep G2 Lane 4: A549 Lane 5: DU 145 Cell lysate: Performed by loading 20ug/lane, of cell lysate and Novex®NuPAGE® 12 % Bis-Tris gel.

Immunohistochemistry analysis of Syndecan-2 of paraffin-embedded human hepatocellular carcinoma tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed using S9111-40B diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunofluorescence analysis of Syndecan-2 was done on 90% confluent log phase Caco-2 cells using S9111-40B. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with S9111-40B (2ug/ml) in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (1:400 ) for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI. F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin. Panel d is a merged image showing junctional localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.