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Product details

Synonyms = FLJ32198, MGC10902, p35, UPIIIb

Antibody type = Mouse monoclonal / IgG

Clone = MSVA-736M

Positive control = Urinary bladder: A moderate to strong membranous Upk3b immunostaining should be seen in umbrella cells of the normal urothelium (the staining is usually limited to apical surface membrane of umbrella cells).

Negative control = Colon: Upk3b immunostaining should be absent in all cells of the colon mucosa.

Cellular localization = Cell Surface and cytoplasm

Reactivity = Human

 

Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only

Relevance of Antibody

Uroplakin 3B is a marker for mesothelial cells and umbrella cells.

 

 

Biology Behind

The Uroplakin 3B (Upk3b) protein is coded by the UPK3b gene located at 7q1.23. Upk3b is one out of 5 known uroplakin (Upk) protein particles that cooperatively form apical asymmetrical unit membrane (AUM) plaques which play an important role in the stabilization and  strengthening of epithelial cells that line the bladder. These AUM plaques enable the inner bladder membrane to stretch and prevent urothelial cells from rupturing during bladder distension. Upks are assembled in the endoplasmic reticulum (ER), where they heterodimerize prior to escaping the ER. Upk3b heterodimerizes with Upk1b. Upk heterodimers subsequently form tetramers which then combine as concentric hexameric rings that are packaged into vesicles and trafficked to the cell surface. AUMs and Upk proteins may have a role in mediating membrane permeability and signal transduction events that are involved in the regulation of cell development, activation, growth, and motility.

Staining Pattern in Normal Tissues

Uroplakin 3B staining pattern in Normal Tissues with antibody MSVA-736M (Images shown in our “Normal Tissue Gallery”)

Brain Cerebrum Negative.
Cerebellum Negative.
Endocrine Tissues Thyroid Negative.
Parathyroid Negative.
Adrenal gland Negative.
Pituitary gland Negative.
Respiratory system Respiratory epithelium Negative.
Lung Negative.
Gastrointestinal Tract Salivary glands Negative.
Esophagus Negative.
Stomach Negative.
Duodenum Negative.
Small intestine Negative.
Appendix Negative.
Colon Negative.
Rectum Negative.
Liver Negative.
Gallbladder Negative.
Pancreas Negative.
Genitourinary Kidney Negative.
Urothelium Moderate to strong, membranous Upk3b staining of the apical membrane of umbrella cells.
Male genital Prostate Negative.
Seminal vesicles Negative.
Testis Negative.
Epididymis Negative.
Female genital Breast Negative.
Uterus, myometrium Negative.
Uterus, ectocervix Negative.
Uterus endocervix Negative.
Uterus, endometrium Negative.
Fallopian Tube Negative.
Ovary Negative.
Placenta early Negative.
Placenta mature Negative.
Amnion Moderate to strong, membranous Upk3b staining of the apical membrane of amnion cells.
Chorion Negative.
Skin Epidermis Negative.
Sebaceous glands Negative.
Muscle/connective tissue Heart muscle Negative.
Skeletal muscle Negative.
Smooth muscle Negative.
Vessel walls Negative (faint staining of the muscular wall or perivascular fibrous structures of small vessels can be seen in case of too sensitive staining protocols).
Fat Negative.
Stroma Negative (faint staining of fibrous structures – often perivascular – can be seen in case of too sensitive staining protocols).
Endothelium Negative.
Bone marrow/lymphoid tissue Bone marrow Negative.
Lymph node Negative.
Spleen Negative.
Thymus Negative.
Tonsil Negative.
Remarks Moderate to strong, membranous Upk3b staining of the apical membrane of mesothelial cells of all organs.

These findings are largely consistent with the RNA and protein data described in the Human Protein Atlas (Tissue expression Uroplakin 3B)  All organs with documented Upk1b RNA expression (urinary bladder, kidney, prostate, gallbladder, stomach, placenta, fallopian tube, uterine cervix, tonsil) with the only exception of smooth muscle are IHC positive for MSV-736M. Given the immediate vicinity of smooth muscle to multiple Upk1b positive tissues, smooth muscle RNA positivity may represent a contamination artifact.

 

Positive control = Urinary bladder: A moderate to strong membranous Upk3b immunostaining should be seen in umbrella cells of the normal urothelium (the staining is usually limited to apical surface membrane of umbrella cells).

Negative control = Colon: Upk3b immunostaining should be absent in all cells of the colon mucosa.

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

According to the TCGA data, Upk3b expression is most commonly seen in urothelial carcinomas and in ovarian cancers. Rarely, it can also be found in other tumors.

The TCGA findings on Uroplakin 3B RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

 

 

Cancer tissue gallery

Compatibility of Antibodies

No data available at the moment

Protocol Recommendations

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-736M 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.

 

Potential Research Applications

  • The prevalence and clinical significance of Upk3b expression in cancer is unknown.
  • The potential diagnostic utility of Upk3b immunostaining needs to be further evaluated.

 

Evidence for Antibody Specificity in IHC

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-736M considerable discrepancy exist as compared to 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 Uroplakin 3B) . In concordance with MSVA-736M IHC data, RNA expression is seen in the urinary bladder, but other tissues with high RNA expression such as esophagus, lung, salivary glands, adipose tissue, testis, vagina, fallopian tube, ovary or the prostate do not show any IHC staining. In addition, MSVA-736M IHC staining observed in amnion cells is not reflected by RNA expression data. This discrepancies are likely to be be due to a contamination of RNA from many organs by (Upk3b expressing) mesothelial cells lining their peritoneal surface of examined tissues. Amnion cells may constitute a too small subpopulation of placenta cells, that Upk3B expression may have been masked by an overwhelming majority of Upk3B negative other cells.

Comparison of antibodies: True expression of Upk3b and correct membranous staining by MSVA-736M of mesothelial, umbrella, and amnion cells is confirmed by comparable staining obtained by a second, independent, commercially available anti-Upk3b antibody, termed “validation antibody”. Independence of the validation antibody is demonstrated by its prominent nuclear staining of numerous tissues such as the skin and the testis, that were not stained by MSVA-736M. These stainings were therefore considered antibody-specific cross-reactivities of the validation antibody.

 

 

Normal tissue gallery