All lanes : Anti-Frizzled 4 antibody [22HCLC] (ab277797) at 2 µg/ml

Lane 1 : HCT 116 cell extract
Lane 2 : HCT116 membrane extract
Lane 3 : T47D cell extract
Lane 4 : T47D membrane extract

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) Superclonal HRP conjugate at 1/2500 dilution

Predicted band size: 60 kDa

Immunohistochemistry analysis of FZD4 showing staining in the cytoplasm of paraffin-embedded human kidney 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 minutes. Following antigen retrieval, tissues were blocked in 3% H₂O₂-methanol for 15 minutes at room temperature, washed with ddH₂O and PBS, and then probed with a FZD4 Recombinant Rabbit Multiclonal Antibody (ab277797) diluted in 3% BSA-PBS at a dilution of 1/100 for 1 hour at 37°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 was performed on fixed and permeabilized T-47D cells for detection of FZD-4 using Anti-FZD-4 Recombinant Rabbit Multiclonal Antibody (ab277797, 2 μg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal^™ Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Panel a) shows representative cells that were stained for detection and localization of FZD-4 protein (green), Panel b) is stained for nuclei (blue) using SlowFade^® Gold Antifade Mountant with DAPI. Panel c) represents cytoskeletal F-actin staining using Alexa Fluor^® 555 Rhodamine Phalloidin (1/300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of FZD-4. Panel e) represents control cells with no primary antibody to assess background.