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Product details

Synonyms = Bone proteoglycan II; CSCD; Dermatan sulphate proteoglycans II (DSPG2); PG40; PGII; PGS2; Proteoglycan core protein; SLRR1B; Small leucine rich protein 1B

Antibody type = Recombinant Rabbit monoclonal / Rabbit IgG

Clone = MSVA-537R

Positive control = Fallopian tube: A strong staining should be seen in the stroma while epithelial cells remain negative.

Negative control = Fallopian tube: Epithelial cells should not show any staining.

Cellular localization = Secreted

Reactivity = Human

 

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

Relevance of Antibody

Decorin is an extracellular matrix protein with multiple functions.

Biology Behind

Decorin is a proteoglycan that weights 90 – 140 kDa and which is coded by the DCN gene on chromosome 12q21.33. Decorin is a member of the small leucine-rich proteoglycan (SLRP) family and represents a significant molecule of the extracellular matrix. It is mainly expressed by fibroblasts and myofibroblasts. Decorin is closely related to biglycan, another component of connective tissue involved in matrix assembly. Decorin appears to influence fibrillogenesis but also interacts with multiple signaling proteins such as fibronectin, thrombospondin, the complement component C1q, and transforming growth factor-beta (TGF-beta) as well as with various receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), Met, and VEGFR. Through these interactions decorin exerts a multitude of onco suppressive functions including growth inhibition, tumor cell mitophagy, and angiostasis. Decorin also has a pro-inflammatory role through modulation of macrophage function and cytokine secretion. Decorin is thus considered a possible therapeutic target for solid malignancies.

Staining Pattern in Normal Tissues

Decorin immunostaining is seen in the stroma of most normal tissues although its quantity is variable. Particularly high levels of staining can be seen along smooth muscle cells, in the myometrium, the stroma of the placenta, endometrium, and the ovary. An intracellular decorin immunostaining occurs in decidua cells (strong, in all cells) and can also be seen in heart muscle and – rarely – in epithelial cells, potentially in case of a focal tissue damage. Squamous epithelial cells – mainly in the granular layer – can show a weak to moderate cytoplasmic staining.

The findings described above are this consistent with the RNA data described in the Human Protein Atlas (Tissue expression Decorin)

 

Positive control = Fallopian tube: A strong staining should be seen in the stroma while epithelial cells remain negative.

Negative control = Fallopian tube: Epithelial cells should not show any staining.

 

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

A decorin immunostaining of variable intensity can be seen in the stroma of cancers from various different entities. More rarely, decorin expression can also occur in the cytoplasm of tumor cells. It has been suggested that a low level of decorin staining in the tumor stroma may be linked to patient prognosis.

The TCGA findings on Decorin 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-537R 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-537R 1:150 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-537R 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-537R 1:150 for 20 minutes at 36°C, secondary antibody (anti-rabbit 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 clinical significance of decorin interactions is not clarified yet.
  • The prognostic impact of decorin immunostaining of cancer cells and tumor stroma is unclear.

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: Given the abundant expression of decorin in various tissues, the protein is not well suited for orthogonal validation. The comparison of the MSVA-537R immunostaining data with 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 Decorin) revealed a good concordance, however. In concordance with RNA expression analysis, decorin immunostaining was much less abundant in  skeletal muscle than in smooth muscle and in heart muscle, and particularly low in lymphatic tissues, the brain, the kidney, and the pancreas.

 

Comparison of antibodies: True expression of decorin in the stroma and occasionally the cytoplasm of specific tissues and cell types is corroborated by comparison with a second commercially available independent antibody (termed “validation antibody”) resulting in identical staining patterns. 

 

Independence of the two antibodies used for validation is documented by an additional cytoplasmic staining in stomach, pancreas, and seminal vesicles which was only seen by the validation antibody but not by MSVA-537R.

 

 

 

 

 

Normal tissue gallery