Product details
Synonyms = caspase 3,CPP32,CPP32B,SCA-1
Antibody type = Recombinant Rabbit monoclonal / IgG
Clone = HMV307
Positive control = Stomach: At least a moderate caspase-3 positivity should be seen in surface epithelial cells.
Negative control = Stomach: Deep gastric glands and muscular cells must be caspase-3 negative.
Cellular localization =Intracellular
Reactivity = Human
Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only
Relevance of Antibody
Caspase-3 is a multifunctional protein with a role in apoptosis regulation.
Biology Behind
Caspase-3 is (in its inactive form) a 32 kD protein which is coded by the CASP3 gene at 4q33-q35.1. Caspase-3 is one of 18 members of the Cysteine-ASPartic proteASES (cysteine proteases) family which share the capacity to mediate the cleavage of specific target proteins. All caspases are all initially produced as inactive procaspases. They can be activated by a wide range of specific internal and/or external signals. Caspase 3 belongs to the subgroup of effector/executioner caspases of the apoptotic subfamily of caspases. Caspase 3 cleave proteins that contain the Asp-Glu-Val-Asp (DEVD) sequence motif. During apoptosis, activated caspase-3 cleaves a multitude of substrates that lead to typical morphological changes in apoptotic cells. For example, caspase-3-mediated cleavage of “inhibitor of caspase-activated DNAse” (ICAD) results in the activation of “Caspase-activated DNAse” (CAD) with subsequent chromatin condensation and DNA fragmentation. In addition to its role as an “executioner of cell death”, caspase-3 can also affect the survival, proliferation, and differentiation of both normal and malignant cells and tissues by both “non-autonomous” and “cell autonomous” mechanisms. A role of caspase-3 is assumed in many diseases including immunologic disease, infectious disease, cancer, and neurological disorders. For example, caspase-3 is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer’s disease.
Staining Pattern in Normal Tissues
Images describing the Caspase-3 staining pattern in normal tissues obtained by the antibody HMV307 are shown in our “Normal Tissue Gallery”.
| Brain | Cerebrum | Weak cytoplasmic caspase-3 positivity of glia cells. |
| Cerebellum | ||
| Endocrine Tissues | Thyroid | Negative. |
| Parathyroid | Faint cytoplasmic caspase-3 positivity of epithelial cells. | |
| Adrenal gland | Weak cytoplasmic caspase-3 positivity of a subset of epithelial cells. | |
| Pituitary gland | Weak to moderate cytoplasmic caspase-3 positivity of epithelial cells of the adenohypophysis. | |
| Respiratory system | Respiratory epithelium | Variable, weak to strong cytoplasmic caspase-3 positivity of respiratory epithelial cells. |
| Lung | Negative. | |
| Gastrointestinal Tract | Salivary glands | Moderate cytoplasmic caspase-3 positivity of few inflammatory cells. |
| Esophagus | Weak cytoplasmic caspase-3 staining of squamous epithelial cells of the lower half of the epidermis. | |
| Stomach | Moderate to strong cytoplasmic caspase-3 positivity of epithelial cells of the surface epithelium. Gastric glands are negative. | |
| Duodenum | Moderate to strong cytoplasmic caspase-3 positivity of surface epithelial cells. Brunner glands are caspase-3 negative. | |
| Small intestine | Strong cytoplasmic caspase-3 positivity of epithelial cells. | |
| Appendix | Moderate cytoplasmic caspase-3 positivity of epithelial cells. Variable (weak to strong) caspase-3 positivity of lymphocytic cells. | |
| Colon | Moderate cytoplasmic caspase-3 positivity of epithelial cells. | |
| Rectum | Moderate cytoplasmic caspase-3 positivity of epithelial cells. | |
| Liver | ||
| Gallbladder | Weak cytoplasmic caspase-3 positivity of epithelial cells. | |
| Pancreas | Faint cytoplasmic caspase-3 positivity of many epithelial cells. | |
| Genitourinary | Kidney | Very faint cytoplasmic caspase-3 positivity of some tubular cells. |
| Urothelium | Moderate to strong cytoplasmic caspase-3 staining of urothelial cells. Staining is weakest in the basal cell layers. | |
| Male genital | Prostate | Negative. |
| Seminal vesicles | Moderate cytoplasmic caspase-3 positivity of epithelial cells. | |
| Testis | Weak cytoplasmic caspase-3 positivity of spermatogonia and (at a lower level) in spermatocytes. | |
| Epididymis | Faint cytoplasmic caspase-3 positivity of epithelial cells of the caput. | |
| Female genital | Breast | Weak to moderate cytoplasmic caspase-3 positivity of luminal epithelial cells. |
| Uterus, myometrium | Negative. | |
| Uterus, ectocervix | Faint cytoplasmic caspase-3 staining of suprabasal squamous epithelial cells. | |
| Uterus endocervix | Negative. | |
| Uterus, endometrium | Negative. | |
| Fallopian Tube | Faint cytoplasmic caspase-3 positivity of respiratory epithelial cells. | |
| Ovary | Weak cytoplasmic caspase-3 positivity of corpus luteum cells. | |
| Placenta early | Moderate to strong cytoplasmic caspase-3 positivity of cytotrophoblast cells. | |
| Placenta mature | Moderate to strong cytoplasmic caspase-3 positivity of trophoblast cells. | |
| Amnion | Negative. | |
| Chorion | Negative. | |
| Skin | Epidermis | Faint cytoplasmic caspase-3 staining of squamous epithelial cells of the lower half of the epidermis. |
| Sebaceous glands | ||
| 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 | Variable (weak to strong) caspase-3 positivity of a fraction of cells. |
| Lymph node | Variable (weak to strong) caspase-3 positivity of lymphocytic cells. | |
| Spleen | Variable (weak to strong) caspase-3 positivity of lymphocytic cells. | |
| Thymus | Variable (weak to strong) caspase-3 positivity of lymphocytic cells. | |
| Tonsil | Weak caspase-3 staining of cryptal epithelial cells. Variable (weak to strong) caspase-3 positivity of lymphocytic cells with strongest staining in germinal centres.. | |
| Remarks |
Caspase-3 is ubiquitously expressed. The highest expression levels occur in lymphoid tissues and the gastrointestinal tract. These findings are largely consistent with the caspase-3 RNA data described in the Human Protein Atlas (Tissue expression Caspase-3).
Positive control = Stomach: At least a moderate caspase-3 positivity should be seen in surface epithelial cells.
Negative control = Stomach: Deep gastric glands and muscular cells must be caspase-3 negative.
Staining Pattern in Relevant Tumor Types
Caspase-3 is often expressed in all kinds of cancers.
The TCGA findings on Caspase-3 RNA expression in different tumor categories have been summarized in the Human Protein Atlas.
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 HMV307 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 role of caspase-3 in neurological and immunological disorders, infectious disease, and in cancer needs to be further clarified.
- The diagnostic, prognostic, and predictive relevance of caspase-3 expression in tumors and in preneoplastic disease 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: Considering the ubiquitous nature of caspase-3 expression, orthogonal validation is not well suited for caspase-3 antibodies. However, for the antibody HMV307 specificity is consistent by the strongest staining seen in the lymphatic tissues and the gastrointestinal tract. These tissues also show highest caspase-3 RNA levels in 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 Caspase-3).
Comparison of antibodies: True expression of caspase-3 in all cell types found caspase-3 positive by HMV307 is corroborated by identical stainings obtained by another commercially available independent antibody (termed “validation antibody”).
