Brachyury (HMV321)

Brachyury protein is a transcription factor coded by the T-box transcription factor T (TBXT) gene at chromosome 6q27. The Brachyury protein has a role in defining the midline of bilaterian organisms by regulating the transcription of genes required for posterior mesoderm formation and cellular differentiation. Brachyury has been implicated in the initiation of chordoma, a rare bone tumor of the axial skeleton. Germ line alterations of TBXT have been linked to familial chordoma. Detectable brachyury protein overexpression has been described to occur in about 90% of chordomas. 

Images describing the Brachyury staining pattern in normal tissues obtained by the antibody HMV321 are shown in our “Normal Tissue Gallery”.

Using our protocol, brachyury staining is not seen in any normal tissues, including parathyroid pituitary gland, and the thyroid where minimal. TBXT RNA expression has been described in the Human Protein Atlas (Tissue expression Brachyury). 

Positive control = Chordoma with previously demonstrated brachyury expression. 

Negative control = Normal kidney: Brachyury staining must be completely absent in all cells (any other normal tissue could be used instead).

Normal tissue gallery

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 HMV321 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• The prevalence and clinical significance of brachyury expression in other (non-chordoma) cancers is not known.
• The function of the brachyury protein needs to be further investigated.
• Considering its absence in normal tissues, the brachyury protein has been suggested as a potential therapeutic target protein.

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 HMV321 specificity is supported by consistency of the immunostaining data with RNA expression data from three independent RNA screening studies, including 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 Brachyury). The complete lack of brachyury Immunostaining in all normal tissues by using a protocol that leads to consistent high-intensity brachyury staining by HMV321 in chordomas is consistent with existing RNA data. RNA expression analyses had identified only very low levels of brachyury RNA expression (<3 nTPM) in only three normal organs (parathyroid, hypophysis, thyroid). The consistent lack of brachyury IHC positivity in these organs suggests that these expression levels are below the threshold for detectability by IHC. 

Comparison of antibodies: True expression of brachyury in chordoma cells with documented brachyury immunostaining by HMV321 is validated by an identical staining obtained by a second, independent commercially available brachyury antibody, termed “validation antibody”.

Normal tissue gallery