710731

antibody from Invitrogen Antibodies

Targeting: FZD4 CD344, EVR1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710731

Provider

Invitrogen Antibodies

Product name

FZD4 Recombinant Polyclonal Antibody (22HCLC)

Antibody type

Polyclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

22HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on cell extracts (30 µg lysate) of HCT 116 (Lane 1), membrane extract of HCT116 (Lane 2), T47D (Lane 3), membrane extract of T47D (Lane 4). The blots were probed with Anti-FZD4 Recombinant Rabbit Polyclonal Antibody (Product # 710731, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 60 kDa band corresponding to FZD4 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on fixed and permeabilized T-47D cells for detection of FZD-4 using Anti- FZD-4 Recombinant Rabbit Polyclonal Antibody (Product # 710731, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 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 (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 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.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

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 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 with a FZD4 Recombinant Rabbit Polyclonal Antibody (Product # 710731) diluted in 3% BSA-PBS at a dilution of 1:100 for 1 hour at 37ºC 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.