Western blot analysis of GATA3 was performed by loading 15 ug of SH-SY5Y whole cell lysate (WCL), SH-SY5Y nuclear extract (NE) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked for at least 1 hour. The membrane was probed with GATA3 polyclonal antibody at a dilution of 1/1000 overnight at 4 degree C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody for 1 hour. Chemiluminescence was performed for band detection
Immunohistochemistry analysis of GATA3 showing staining in the nucleus of paraffin-embedded human tonsil tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM 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 GATA3 polyclonal antibody diluted in 3% BSA-PBS at a dilution of 1/200 overnight at 4 degree 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 DAB. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting
Immunofluorescent analysis of GATA3 (green) in MCF7 (left panel) and SH-SY5Y cells (right panel). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with GATA3 polyclonal antibody at a dilution of 1/50 for at least 1 hour at room temperature, washed with PBS, and incubated with a goat anti-rabbit IgG secondary antibody (green) for 30 minutes at room temperature. Cells were counterstained with phalloidin (F-actin, red) and Hoechst 33342 (Nucleus, blue). Cells were viewed at 20x magnification
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