PAI2 (HMV330)

Plasminogen activator inhibitor-2 (placental PAI, SerpinB2, PAI-2) is a serine protease inhibitor of the serpin superfamily coded by the SerpinB2 gene on chromosome 18.q22.1. It acts as a coagulation factor that irreversibly inactivates tissue plasminogen activator and urokinase. PAI2 exists as a 60-kDa extracellular glycosylated form and a 43-kDa intracellular form. PAI2 protein is normally not detectable in adult plasma. The protein is produced at large quantities in the placenta which explains that PAI2 is detectable in blood only during pregnancy. PAI2 may thus contribute to the increased rate of thrombosis during pregnancy. PAI2 can bind to multiple intracellular and extracellular proteins. For example, it was suggested that PAI2 may activate p53 and stabilize p21. Macrophage derived PAI2 plays a role in inflammatory responses and infections, potentially in downregulating T cells that secrete IgG2c and interferon type II. Although glycosylated extracellular PAI2 regulates fibrinolysis, it remains unclear whether this is the main or entire role of PAI2. PAI2 is predominantly intracellular but specific intracellular roles for PAI2 have not yet been identified. PAI2 may play a complex role in cancer. Both tumor-promoting and tumor-inhibiting effects have been described.

Images describing the PAI2 staining pattern in normal tissues obtained by the antibody HMV330 are shown in our “Normal Tissue Gallery”.

PAI2 staining in normal tissues is typically cytoplasmic and nuclear. The strongest staining occurs in trophoblastic cells and in amnion cells of the placenta as well as in decidua cells of the endometrium (during pregnancy). The PAI2 staining pattern in squamous epithelium is complex and typically limited to specific maturation steps/cell layers. In most non-keratinizing squamous epithelia, a weak to strong cytoplasmic and nuclear PAI2 staining occurs in the upper half or upper two-thirds of the epithelium while basal and suprabasal layers are PAI2 negative. In the epidermis, at least in some samples, PAI2 staining can also predominate in the bottom 2/two-thirdof the squamous epithelium including the basal cell layer. A moderate to strong PAI2 staining also occurs in a fraction of cells of corpuscles of Hassall‘s of the thymus, a small fraction of epithelial cells of the pancreas, a subset of epithelial cells from excretory ducts (not in all samples), and (weakly) few dispersed corpus luteum cells of the ovary. PAI2 staining also occurs in a small fraction of monocytes / macrophages. PAI2 staining is absent in all mesenchymal tissues, cerebrum, cerebellum, lymphocytes, sebaceous and ekkrine glands of the skin, urothelium, mucosa of the stomach, duodenum, ileum, appendix, colon, rectum and gallbladder, liver, Brunner gland of the duodenum, kidney, prostate, seminal vesicle, epididymis, testis, respiratory epithelium and glands of bronchi and sinus paranasales, lung, breast, endocervix, endometrium, fallopian tube, adrenal gland, parathyroid gland, and pituitary gland.

These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression PAI2). 

Positive control = Tonsil: A strong cytoplasmic and nuclear PAI2 staining must be seen in a large fraction of squamous epithelial cells. 

Negative control = Tonsil: Lymphocytes must be PAI2 negative (few monocytes/macrophages may show PAI2 staining).

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 HMV330 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 diagnostic and prognostic relevance of PAI2 IHC in tumors and in preneoplastic disease is unresolved.
• The function/role of intracellular PAI2 has not yet been identified. 
• The role of PAI2 in cancer is unclear.

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 HMV330 specificity is supported by the strong concordance with RNA expression data derived 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 PAI2). In agreement with these data, PAI2 immunostaining was observed in all tissues covered by squamous epithelium (esophagus, tonsil cervix uteri, skin), salivary glands, the placenta, and in the bone marrow. PAI2 positivity by HMV330 was, however, also detectable in several tissues without documented PAI2 RNA expression (decidua cells, amnion cells, corpuscles of Hassall‘s of the thymus, a small fraction of epithelial cells of the pancreas, few dispersed corpus luteum cells of the ovary). Some of these tissues without previously documented PAI2 RNA expression but PAI2 positivity by HMV330 had only very few positive cells that were probably not detected in RNA studies (corpuscles of Hassall‘s of the thymus, a small fraction of epithelial cells of the pancreas). Other cell types had not previously been specifically analyzed on the RNA level (decidua cells, amnion cells, corpus luteum cells). For some tissues with significant but low levels of PAI2 RNA expression (breast, fallopian tube, appendix, urinary bladder, fat), a significant PAI2 immunostaining by HMV330 was not observed in 8 analyzed samples.

Comparison of antibodies: True expression of PAI2 in all cell types found to be PAI2 positive by HMV330 is corroborated by a confirmation of these staining’s by another commercially available independent antibody (termed “validation antibody”). This applies to cell types from organs that were considered PAI2 RNA negative such as corpuscles of Hassall‘s of the thymus, a small fraction of epithelial cells of the pancreas, as well as to cell types for which PAI2 RNA data were not available (decidua cells, amnion cells, corpus luteum cells). Independence of the validation antibody is documented by various stainings (hepatocytes, renal tubuli, testis, thyroid) that were only observed by the validation antibody but not by HMV330.

Normal tissue gallery