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Product details

Synonyms = Succinate dehydrogenase complex flavoprotein subunit A , CMD1GG , FP , PGL5 , SDH1 , SDH2 , SDHF

Antibody type = Recombinant Rabbit monoclonal / IgG

Clone = HMV336

Positive control =  Appendix: A strong SDHA staining should be seen in epithelial cells and a weak to moderate staining should occur in lymphocytic cells.

Negative control = SDHA staining should be absent in all cells of a tumor with known SDHA expression loss.

Cellular localization = Intracellular

Reactivity = Human

 

 

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

Relevance of Antibody

SDHA is a critical protein component of the SDH complex.

Biology Behind

Succinate dehydrogenase complex iron sulfur subunit A (SDHA) is coded by the SDHA gene at chromosome 5p15.33. SDHA forms the succinate dehydrogenase protein complex II together with SDHB, SDHC and SDHD. Within this complex, SDHA is linked to SDHB at the catalytic end of the SDH protein complex which protrudes into the mitochondrial matrix while SDHC and SDHD are hydrophobic and attach the SDH complex to the inner mitochondrial membrane. SDH plays a pivotal role in both the citric acid cycle and the respiratory chain. It catalyzes the conversion of succinate to fumarate in the citric acid cycle downstream of IDH2.  A loss of enzyme activity results in accumulation of metabolic intermediates similarly as under hypoxic conditions. Such a “pseudohypoxia” results in HIF-1 pathway activation and may induce a metabolic shift toward aerobic glycolysis. SDHA germline mutations can cause pheochromocytoma, paraganglioma, gastrointestinal stromal tumor (GIST), pituitary adenoma and renal cell carcinoma. SDHA immunohistochemistry (IHC) can be used to identify SDHA deficiency while SDHB IHC is a surrogate tool for assessing the entire SDH complex (SDHA, SDHB, SDHC, SDHD) including the activity of its assembling factor SDHAF2.

Staining Pattern in Normal Tissues

Images describing the SDHA staining pattern in normal tissues obtained by the antibody HMV336 are shown in our “Normal Tissue Gallery”.

Brain Cerebrum Rather weak granular cytoplasmic SDHA staining of cells.
Cerebellum Rather weak granular cytoplasmic SDHA staining of cells.
Endocrine Tissues Thyroid Weak granular cytoplasmic SDHA staining of epithelial cells.
Parathyroid Weak to strong granular cytoplasmic SDHA staining of epithelial cells. Marked heterogeneity between cells and cell groups.
Adrenal gland Strong granular cytoplasmic SDHA staining of epithelial cells.
Pituitary gland Strong granular cytoplasmic SDHA staining of epithelial cells. Moderate granular cytoplasmic SDHA staining of pituicytes and fibres.
Respiratory system Respiratory epithelium Marked granular cytoplasmic SDHA staining of epithelial cells, predominately in the sub-apical compartment.
Lung SDHA staining is largely absent in alveocytes. SDHA staining is prominent in macrophages and bronchiole..
Gastrointestinal Tract Salivary glands Granular cytoplasmic SDHA staining is strong in excretory ducts and only faint in glandular cells.
Esophagus Moderate granular cytoplasmic SDHA staining, predominantly of basal and suprabasal squamous epithelial cells.
Stomach A granular cytoplasmic SDHA staining occurs in all cells but it is strongest in stomach glands, especially in parietal cells.
Duodenum Strong granular cytoplasmic SDHA staining occurs in all epithelial cells. The staining predominates in the sub-apical compartment and it is stronger in villi than in crypts. Weak to moderate SDHA staining of  Brunner gland cells.
Small intestine Strong granular cytoplasmic SDHA staining of all epithelial cells. It predominates in the sub-apical compartment and it is slightly stronger in villi than in crypts.
Appendix Strong granular cytoplasmic SDHA staining of all epithelial cells.
Colon At least a moderate granular cytoplasmic SDHA staining of all epithelial cells.
Rectum At least a moderate granular cytoplasmic SDHA staining of all epithelial cells.
Liver Strong granular cytoplasmic SDHA staining of cells, especially in hepatocytes.
Gallbladder Weak to moderate granular cytoplasmic SDHA staining of epithelial cells, predominately in the sub-apical compartment.
Pancreas Granular cytoplasmic SDHA staining of all epithelial cells. It is weakest in islet cells and strongest in some small excretory ducts.
Genitourinary Kidney Intense granular cytoplasmic SDHA staining of tubuli and cells of collecting ducts while staining is weak in glomeruli.
Urothelium Weak granular cytoplasmic SDHA staining of urothelial cells (all layers).
Male genital Prostate Rather weak granular cytoplasmic SDHA staining – somewhat stronger in epithelial than in stromal cells.
Seminal vesicles Strong granular cytoplasmic SDHA staining in most epithelial cells.
Testis Granular cytoplasmic SDHA staining is strongest in maturing germ cells.
Epididymis Moderate to strong granular cytoplasmic SDHA staining, predominately in the sub-apical compartment, in epithelial cells of the cauda. Faint granular cytoplasmic SDHA epithelial cell staining in the corpus.
Female genital Breast Moderate to strong granular cytoplasmic SDHA staining of epithelial cells.
Uterus, myometrium Weak to moderate granular cytoplasmic SDHA staining of muscle cells.
Uterus, ectocervix Weak to moderate granular cytoplasmic SDHA staining, largely limited to suprabasal squamous epithelial cells.
Uterus endocervix Moderate to strong granular cytoplasmic SDHA staining of epithelial cells. SDHA staining is less intense in stromal cells.
Uterus, endometrium Weak to moderate granular cytoplasmic SDHA staining of epithelial cells.
Fallopian Tube Weak to moderate granular cytoplasmic SDHA staining of epithelial cells.
Ovary Moderate to strong granular cytoplasmic SDHA staining of stromal cells. Moderate SDHA staining of corpus luteum cells.
Placenta early Weak granular cytoplasmic SDHA staining of throphoblast cells.
Placenta mature Weak granular cytoplasmic SDHA staining of throphoblast cells.
Amnion Weak to moderate/strong granular cytoplasmic SDHA staining of amnion cells.
Chorion Moderate granular cytoplasmic SDHA staining of chorion cells.
Skin Epidermis Weak to moderate granular cytoplasmic SDHA staining, predominantly of basal and suprabasal squamous epithelial cells.
Sebaceous glands
Muscle/connective tissue Heart muscle Strong granular cytoplasmic SDHA staining.
Skeletal muscle Moderate to strong granular cytoplasmic SDHA staining.
Smooth muscle Weak granular cytoplasmic SDHA staining.
Vessel walls Weak to moderate granular cytoplasmic SDHA staining.
Fat Lack of unequivocal SDHA staining.

 

Stroma Granular cytoplasmic SDHA staining of Variable intensity (mostly weak).
Endothelium Granular cytoplasmic SDHA staining of Variable intensity (mostly weak).
Bone marrow/ lymphoid tissue Bone marrow Granular cytoplasmic SDHA staining is only faint, if at all visible.
Lymph node Weak to moderate granular cytoplasmic SDHA staining of lymphocytes.
Spleen Weak to moderate granular cytoplasmic SDHA staining of a fraction of inflammatory cells.
Thymus Weak to moderate granular cytoplasmic SDHA staining of lymphocytes.
Tonsil Weak to moderate granular cytoplasmic SDHA staining of squamous epithelial cells and of lymphocytes.
Remarks In principle, all cells do show a granular cytoplasmic SDHA staining. The staining intensity depends on the cell type and perhaps also to the functional state of the cells.

 

These findings are largely consistent with the TIGIT RNA data described in the Human Protein Atlas (Tissue expression SDHA). SDHA staining by HMV336 is most prominent in heart and skeletal muscle, liver, kidney, parathyroid gland, adrenal gland, and gastrointestinal epithelium but also occurs – often at lower levels – in a very broad range of other tissues.

 

Positive control =  Appendix: A strong SDHA staining should be seen in epithelial cells and a weak to moderate staining should occur in lymphocytic cells.

Negative control = SDHA staining should be absent in all cells of a tumor with known SDHA expression loss.

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

A variable level of SDHA expression occurs in virtually all tumors except those with an SDH deficiency. According to RNA data of the TCGA database, the level of SDHA expression is highly prognostic in renal cell carcinomas.

The TCGA findings on SDHA 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 HMV336 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.

Potential Research Applications

  • The prognostic relevance of SDHA expression in tumors and in preneoplastic disease needs to be investigated. 
  • The predictive relevance of SDHA expression in tumors is unknown. 
  • The role of SDHA expression levels and of SDHA mutation in non-neoplastic disease is unknown.

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 HMV336 specificity is supported by the good concordance 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 SDHA). SDHA positivity by HMV336 is particularly strong in heart and skeletal muscle, liver, kidney, parathyroid gland, adrenal gland, and gastrointestinal epithelium, all tissues with a particularly high SDHA RNA expression. The value of orthogonal limitation is limited, however, in case of ubiquitously expressed proteins.

 

Comparison of antibodies: True expression of SDHA in all cell types found SDHA positive by HMV336 is corroborated by identical relative levels of staining intensity obtained by another commercially available independent antibody (termed “validation antibody”) although the staining by the validation antibody was always less intense. 



 

 

 

 

Normal tissue gallery