GATA3 (MSVA-450M)

GATA3 gene at 10p14 consists of 8 exons and codes for GATA3, variant 1, and GATA3, variant 2, proteins of 48 KDa that which differ in one amino acid and may have identical functions. GATA3 is characterized by two conserved zinc finger domains, which bind DNA at the nucleotide sequence GATA. Like other proteins of the GATA family (GATA1-6), GATA3 is a transcription factor that regulates various cellular programs during embryogenesis and differentiation in several organ systems, including parathyroid gland, kidney, breast epithelium, urothelium, and the nervous system. A key role has been identified in T cell development, where GATA3 is the downstream of Notch in differentiation of T-helper cells and important for allergic and humoral immune responses. A role of GATA3 has also been demonstrated for tumor development and progression. GATA3 is frequently found mutated in breast cancer, and downregulation of the protein has been linked to poor prognosis of the disease.

GATA3 staining pattern in Normal Tissues with antibody MSVA-450M (images are shown in our “Normal Tissue Gallery”)

The findings obtained by MSVA-450M almost perfectly match the RNA data summarized in the Human Protein Atlas (Tissue expression GATA3)  and identify the cell types responsible for RNA expression. The only protein atlas findings which could not be corroborated was GATA3 expression in the vagina. The validation experiments performed with MSVA-450M had suggested that GATA3 expression occurs in all keratinized squamous epithelia but not in non-keratinized squamous epithelia.

Suggested positive tissue control:
-Kidney: A moderate to strong nuclear staining reaction should be seen in a fraction of collecting duct cells and podocytes in glomeruli.
-Tonsil: The vast majority of T helper cells (Th2) in the T-zones should show a weak to moderate nuclear staining. 

Suggested negative tissue control:
-Kidney: Staining should be absent in proximal and distal tubuli as well as in blood vessels.
-Tonsil: Staining should be absent in B-cells.

Normal tissue gallery

Detectable expression of GATA3 is primarily seen in breast cancer and in urothelial carcinomas. Other tumors for which GATA3 expression has been reported include renal carcinoma, lung cancer and testicular tumors. At low frequency, GATA3 expression can occur in additional tumor entities. 

The TCGA findings on GATA3 RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply MSVA-450M at a dilution of 1:80 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

Agilent / Dako – Autostainer Link 48

Pretreatment in PT-Link for 30 minutes at 95°C (pH high); FLEX peroxidase blocking for 5 minutes (room temperature), MSVA-450M 1:80 for 20 minutes (room temperature), FLEX+ mouse/rabbit (LINKER) for 15 minutes (room temperature), horseradish peroxidase (HRP) for 20 minutes (room temperature), FLEX DAB+Sub-Chromo for 10 minutes (room temperature), FLEX hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, and a longer incubation time of FLEX+LINKER result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Leica – BOND RX

Dewax at 72°C for 30 seconds; Pretreatment in Bond Epitope Retrieval Solution (ER2 – EDTA pH9) for 20 minutes at 100°C; Peroxidase blocking for 5 minutes (room temperature), MSVA-450M 1:80 for 15 minutes (room temperature), Post primary (rabbit anti mouse) for 8 minutes (room temperature), Polymer (goat anti rabbit) for 8 minutes (room temperature), mixed DAB refine for 10 minutes (room temperature), hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of Post primary and or the Polymer result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

-Potential pitfalls

GATA3 is used as a marker for urinary bladder cancer. However, squamous cell carcinoma of the cervix uteri can also express GATA3 at low levels. This may cause misinterpretation as “urothelial carcinoma” in case of a cervical carcinoma infiltrating the urinary bladder.

• Because of partly controversial data, the diagnostic utility of GATA3 IHC should be investigated in a large cohort of tumors from different entities.
• The prognostic role of GATA3 expression in tumor types that are positive in only a fraction of cases is unclear.
• GATA3 is one of the three genes mutated in >10% of breast cancers (Cancer Genome Atlas) and plays a role in estrogen and androgen receptor signaling. Further studies are needed to elucidate the role, if any, of GATA3 in the development of breast cancer.
• GATA3 delineates important subgroups of inflammatory cells of which the exact biological and clinical role is not fully understood. GATA3 is thus an important component of antibody panels for use in multicolor immunofluorescence analyses.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody MSVA-450M specificity is documented by the strong concordance of the immunostaining with RNA expression data derived from the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project which are all summarized in the Human Protein Atlas (Tissue expression GATA3). Immunostaining by using MSVA-450M was only detected in organs with documented RNA expression. The only RNA finding which could not be corroborated was GATA3 expression in the vagina. It appears possible that the RNA analyses erroneously included GATA3 positive keratinized squamous epithelium from the vaginal orifice. 

Comparison of antibodies: Specific GATA3 staining of MSVA-450M is supported by a staining of the same cell types by an independent commercial “validation antibody”. This applies for urothelium, squamous epithelium of the skin, hair follicles, sebaceous glands, parathyroid gland, trophoblastic, chorion and amnion cells of the placenta, collecting ducts and glomerular podocytes of the kidney, seminal vesicle epithelium, luminal cells of breast glands, and a fraction of lymphocytes, most prominently in the thymus (comparative images shown below). Because the sensitivity of the validation antibody is lower than for MSVA-450M at the selected protocol, the validation antibody exhibits only a very faint staining in tall columnar cells and basal cells of the epididymis, basal cells in the prostate, and glandular cells of salivary glands. True GATA3 expression in these tissues is further corroborated by a staining seen by the antibody CAB016217 used for protein analysis in the human protein atlas. Images there show staining in the epididymis , prostate basal cells  and in salivary glands.

A weak cytoplasmic GATA3 staining that can be seen in gastric glands and goblet cells of respiratory epithelium, most likely represents non-specific background staining.

Antibody Comparison: MSVA-450M vs another commercially available GATA3 antibody called “Validation Antibody”

Normal tissue gallery