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Product details

Synonyms = UP1A; UPIA; UPKA; TSPAN21

Antibody type = Mouse monoclonal / IgG

Clone = MSVA-735M

Positive control =Urinary bladder:  A strong membranous and cytoplasmic Upk1a immunostaining should be seen in the urothelium (the staining can be limited to the top 1/3 or 2/3 of the urothelium).

Negative control =Colon:  Upk1a immunostaining should be absent in all cells of the colon mucosa.

Cellular localization = Cell surface

Reactivity = Human

 

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

Relevance of Antibody

Uroplakin 1A is marker for urothelium and urothelial tumors.

Biology Behind

The Uroplakin 1A (Upk1a) protein is coded by the UPK1b gene located at 19q13.12. Upk1a is one out of 5 known uroplakin (Upk) protein particles that cooperatively form apical asymmetric 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. Upk1a heterodimerizes with Upk2 and Upk1b heterodimerizes with Upk3. Upk heterodimers subsequently form heterotetramers 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 1A staining pattern in Normal Tissues with antibody MSVA-735M (images are 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 A faint/weak/moderate Upk1a staining can occur in some cell layers (middle third) of the squamous epithelium.
Stomach Negative.
Duodenum Negative.
Small intestine Negative.
Appendix Negative.
Colon Negative.
Rectum Negative.
Liver Negative.
Gallbladder Negative.
Pancreas Negative.
Genitourinary Kidney Negative.
Urothelium Upk1a immunostaining is strongest in the urothelium. Here the staining is particularly strong in the top third cell layers including umbrella cells. Staining is less intense or even absent in basal cell layers.
Male genital Prostate A focal weak to moderate UpK1a staining can occur in case of squamous metaplasia in prostatic glands or ducts.
Seminal vesicles A moderate to strong focal UpK1a staining of individual cells or groups of cells can occasionally be seen.
Testis Negative.
Epididymis Negative.
Female genital Breast Negative.
Uterus, myometrium Negative.
Uterus, ectocervix A faint/weak/moderate Upk1a staining can occur in some cell layers (middle third) of the squamous epithelium.
Uterus endocervix Negative.
Uterus, endometrium Negative.
Fallopian Tube Negative.
Ovary Negative.
Placenta early Negative.
Placenta mature Negative.
Amnion Negative.
Chorion Negative.
Skin Epidermis A faint/weak/moderate Upk1a staining can occur in some cell layers (middle third) of the squamous epithelium.
Sebaceous glands Negative.
Muscle/connective tissue Heart muscle Negative.
Skeletal muscle Negative.
Smooth muscle Negative.
Vessel walls Negative.
Fat Negative.
Stroma Negative.
Endothelium Negative.
Bone marrow/ lymphoid tissue Bone marrow Negative.
Lymph node Negative.
Spleen Negative.
Thymus Negative.
Tonsil Negative.
Remarks

 

These findings are largely consistent with the RNA and protein data described in the Human Protein Atlas (Tissue expression Uroplakin 1A) 

 

Positive control =Urinary bladder:  A strong membranous and cytoplasmic Upk1a immunostaining should be seen in the urothelium (the staining can be limited to the top 1/3 or 2/3 of the urothelium).

Negative control =Colon:  Upk1a immunostaining should be absent in all cells of the colon mucosa.

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

A positive Upk1a immunostaining is most commonly seen in urothelial carcinomas. It can also occur in squamous cell  carcinomas and – rarely – in other cancers.

The TCGA findings on Uroplakin 1A 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 9 Target Retrieval Solution buffer. Apply MSVA-735M 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.

 

 

 

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-735M 1:450 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-735M 1:150 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.

 

Roche – Ventana Discovery ULTRA

Pretreatment for 64 minutes at 100°C (pH 8,4); CM peroxidase blocking for 12 minutes (room temperature), MSVA-735M 1:150 for 20 minutes at 36°C, secondary antibody (anti-mouse HQ) for 12 minutes at 36°C, anti-HQ HRP for 12 minutes at room temperature, DAB at room temperature, hematoxylin II at room temperature for 8 minutes, bluing reagent at room temperature for 4 minutes.

These images depict staining results obtained by the protocol described above. It is of note, that the Ventana machines generally require higher antibody concentrations than other commonly used autostainers because the antibodies are automatically diluted during the procedure. Various other protocols can result in an identical result as shown above. 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 secondary antibody and or the anti-HQ HRP result in stronger staining, potentially at the cost of more background staining.

Potential Research Applications

  • The prevalence and clinical significance of Upk1a expression in cancer is unknown.