NFIX (HMV329)

Nuclear factor 1 X-type (NFIX) is a transcription factor protein which is coded by the NFIX gene on chromosome 19p13.13. Besides NFIA, NFIB, NFIC, NFIX is one out of four closely related members of the nuclear factor I (NFI) family of transcription factors. The family members share a particularly high rate of interactions with other transcription factors. They are therefore thought to not only directly regulate the expression of genes but also believed to modulate the function of other transcription factors. NFIX is known to play a role in muscle and central nervous system embryonic development. For example, NFIX has been shown to control the timing of neural differentiation promoting the ongoing growth of the hippocampus and proper memory function. The role of NFIX in other tissues is less intensively studied. Various types of NFIX alterations have been found in tumors. Mechanisms leading to both increased and reduced expression have been found to promote pro-tumorigenic functions, such as increased cell proliferation and migration and disturbed differentiation.

In normal tissues, NFIX is ubiquitously seen in nuclei of all tissues and most cell types. Images describing the NFIX staining pattern in normal tissues obtained by the antibody HMV329 are shown in our “Normal Tissue Gallery”.

These findings are largely comparable to the RNA and protein data described in the Human Protein Atlas (Tissue expression NFIX).

Positive control = Placenta: A moderate to strong nuclear NFIX staining should be seen in stromal cells while trophoblast cells should remain NFIX negative.

Negative control = Placenta: Trophoblast cells should remain NFIX negative while a moderate to strong nuclear NFIX staining should be seen in stromal cells.

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 HMV329 at a dilution of 1:200 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• The role of NFIX in cancer is completely unclear.
• The patterns of NFIX protein expression in cancer have not been explored. 
• The role of NFIX in heart disease is under investigation.
• The role of NFIX in other diseases is not specified yet.

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 HMV329, specificity is consistent by the consistency of the immunostaining data with 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 NFIX). In agreement with HMV329 immunostaining data, NFIX RNA expression predominated in muscle tissues, was high in salivary glands, fallopian tube and the endometrium, and was low or absent in bone marrow, lymphatic tissues, kidney, liver and the testis. However, orthogonal validation is not optimal for proteins that are expressed in all organs.

Comparison of antibodies: True expression of NFIX in all cell types with nuclear NFIX positivity by HMV329 is corroborated by an identical nuclear staining obtained by a commercially available independent second antibody (termed “validation antibody”). Most of all, both antibodies showed several characteristic nuclear staining patterns such as strong staining in muscular tissue, ovarian stroma, epidermis, non-keratinizing squamous epithelium, respiratory epithelium, urothelium, excretory ducts and intercalated ducts of the pancreas, salivary epithelium, prostate epithelial cells, basal cells of the caput epididymis, stroma and Leydig cells of the testis, the lung, basal and luminal epithelial cells of the breast gland, endometrial stroma, fallopian tube, follicular cells of the thyroid, and in pituicytes. Absence of nuclear NFIX staining was confirmed by the validation antibody in umbrella cells, amnion, chorion, and trophoblast cells, lymphocytes, macrophages, most hematopoietic cells of the bone marrow, hepatocytes, Sertoli cells and germ cells of the testis, endometrium glands, corpus luteum, adrenal medullary cells, Purkinje cells of the cerebellum, and in most if not all cells of the adenohypophysis.

Independence of the validation antibody is demonstrated by a high level cytoplasmic cross-reactivity in a broad range of different cell types which was only seen by the  and not by HMV329.

Normal tissue gallery