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- Table of Contents
Plan TRA2A paraffin IHC using the reported cytoplasmic staining in smooth muscle cells (HPA tissue IHC). Compare the observed cell type and compartment with controls, while accounting for the annotated nuclear location (UniProt) and uncertain tissue staining reliability (HPA tissue IHC).
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC); nuclear location (UniProt) | |
| Staining pattern | Cytoplasmic staining is most abundant in smooth muscle cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Heart muscle+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03770) | |
| Caveat | Tissue staining awaits external verification (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | Four isoforms; epitope overlap is undetermined (UniProt) |
The catalog antibody’s IHC-P protocol is accompanied by one published TRA2A IHC protocol (PMC9275993).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A03770) |
| Fixation | Image fixative and duration unreported (datasheet A03770); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-TRA2A, 1:100-1:300 (datasheet A03770) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TRA2A-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression, most abundant in smooth muscle cells. No signal in the no-primary control. |
For paraffin-section IHC, HPA reports predominantly cytoplasmic TRA2A staining, most abundant in smooth muscle cells; cardiomyocytes, kidney tubule cells and pancreatic exocrine cells also score High (HPA: tissue IHC). Interpret this cautiously: the tissue profile is Uncertain, and HPA054018 has Uncertain IHC validation (HPA: tissue IHC; HPA: antibody validation). UniProt places TRA2A in the nucleus and reports no transmembrane segment (UniProt Q13595: subcellular location and topology).
| Strong cytoplasmic staining in smooth muscle cells, with staining in the expected cell populations of a comparison section. | This matches the reported tissue IHC profile; cardiomyocytes, kidney tubule cells and pancreatic exocrine cells are also High (HPA: tissue IHC). It is a provisional positive pattern because the profile and HPA054018 IHC validation are Uncertain (HPA: tissue IHC; HPA: antibody validation). |
| Predominantly membrane-edge staining, with little signal in the expected cells or compartments. | Treat a membrane-only pattern as suspect: TRA2A is annotated as nuclear and has no transmembrane segment (UniProt Q13595: location and topology). Check whether the signal follows section edges or persists when the primary antibody is omitted (general IHC practice). |
| Strong staining in cell populations scored Not detected, such as adrenal glandular cells or bronchial respiratory epithelial cells. | Consider cross-reactivity or endogenous detection activity, especially if expected positive cells do not stain (HPA: tissue IHC; general IHC practice). A Not detected entry applies to the named cell population and does not make every cell in that tissue a negative control (HPA: tissue IHC). |
| A diffuse chromogen haze covers cells and surrounding tissue without clear cell boundaries. | This cannot be scored as a TRA2A cell pattern; compare the no-primary control and inspect blocking, washing and detection background (general IHC practice). HPA's cytoplasmic tissue observation concerns stained cells, not uniform section haze (HPA: tissue IHC). |
| No convincing signal in smooth muscle cells on an otherwise intact section. | The result conflicts with a High-scoring HPA cell population (HPA: tissue IHC). First check the run controls and detection steps, then review the catalog antibody's IHC-P instructions (general IHC practice). HPA's Uncertain reliability prevents calling this specimen TRA2A-negative from one failed stain (HPA: tissue IHC). |
| Tissue IHC evidence and antibody validation | HPA calls the tissue profile Uncertain, pending external verification, and rates HPA054018 IHC Uncertain (HPA: tissue IHC; HPA: antibody validation). Treat agreement with the reported pattern as supporting evidence, not proof of antibody specificity. |
| Compartment differs by assay | HPA tissue IHC describes cytoplasmic expression, while UniProt annotates a nuclear location (HPA: tissue IHC; UniProt Q13595: subcellular location). Keep assay and cell context attached to each observation; these sources do not resolve why the compartments differ. |
| Isoforms and processing | UniProt lists 4 isoforms and a chain spanning residues 2–282, with no signal peptide or propeptide (UniProt Q13595: isoforms and processing). The payload gives no antibody epitope or isoform coverage, so an unexpected pattern cannot be assigned to an isoform. |
| IF/ICC Q: What localisation should be expected? | A: HPA reports mainly nucleoli, with additional nucleoplasm and vesicles; nucleoli and nucleoplasm are Supported, and vesicles are Approved (HPA: subcellular ICC-IF). HPA054018 is ICC Supported (HPA: antibody validation). Apply that evidence to IF/ICC interpretation, not as an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The presumed positive section has no smooth muscle signal. | The IHC run or detection may have failed; smooth muscle cells score High, although the tissue profile is Uncertain (HPA: tissue IHC; general IHC practice). | Check that counterstain and run controls are acceptable, then review the catalog antibody's IHC-P instructions and detection reagents (general IHC practice). Repeat with a documented positive section before scoring the specimen negative. |
| The whole section is brown, including regions without identifiable positive cells. | Diffuse background may reflect nonspecific detection or endogenous enzyme activity rather than cell-specific TRA2A signal (general IHC practice). | Compare a no-primary control, inspect blocking and wash steps, and assess endogenous activity controls appropriate to the chromogen system (general IHC practice). Score only signal that resolves to cells. |
| Adrenal glandular cells or bronchial respiratory epithelial cells stain strongly. | Those named populations are Not detected in HPA tissue IHC; cross-reactivity or detection background is possible, but the HPA profile is Uncertain (HPA: tissue IHC; general IHC practice). | Confirm cell identity and compare the no-primary control with an HPA High cell population in the same run (HPA: tissue IHC; general IHC practice). Avoid interpreting one discrepant tissue as definitive specificity evidence. |
| Signal appears only at section edges or along membranes. | Edge artefact or nonspecific deposition is plausible; UniProt annotates nuclear TRA2A without a transmembrane segment (UniProt Q13595: location and topology; general IHC practice). | Inspect section quality and staining distribution, compare a no-primary control, and repeat on a well-preserved section if the pattern persists (general IHC practice). |
| Nuclear staining appears alongside the reported IHC cytoplasmic pattern. | The sources disagree across assays: UniProt annotates nucleus, HPA tissue IHC describes cytoplasm, and HPA ICC-IF supports nucleoli and nucleoplasm (UniProt Q13595: location; HPA: tissue IHC; HPA: subcellular ICC-IF). | Record nuclear and cytoplasmic staining separately by cell type. Compare positive and no-primary controls, and retain the IHC reliability caveat when interpreting either compartment (HPA: tissue IHC; general IHC practice). |
| Different specimens show different proportions of stained cells. | HPA reports High, Medium, Low and Not detected levels in specified tissue cell populations, with Low tissue RNA specificity overall (HPA: tissue IHC). Those observations do not establish a universal intensity cutoff. | Compare the same cell population across similarly processed sections, document intensity and distribution, and use a consistent run control (general IHC practice). Do not treat staining elsewhere in an HPA-listed tissue as the listed cell's result. |
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Troubleshoot TRA2A staining in paraffin sections by checking retrieval, compartment, controls, and cell specific scoring before interpreting chromogenic signal.
Anti-TRA2A antibodies A03770, A03770-1 and A30486 have IHC images from paraffin-embedded human brain tissue (catalog IHC image captions); A03770 and A30486 also have IF images from HeLa cells (catalog IF image captions).
A03770 and A30486 each have an IHC image from paraffin-embedded human brain tissue and an IF image from HeLa cells (A03770 and A30486 image captions); both list Human and Mouse reactivity (catalog reactivity). A03770-1 has an IHC image from paraffin-embedded human brain tissue, lists IF among its applications, and lists Human, Mouse and Rat reactivity (A03770-1 image caption; catalog applications and reactivity).
Which to pick: For tissue IHC, all three have images from paraffin-embedded human brain tissue (each SKU’s IHC image caption); start within the listed IHC dilutions of 1:100–1:300 for A03770 or A30486, or 1:50–1:200 for A03770-1 (catalog dilutions). For IF/ICC, choose A30486 when ICC is required because it lists both ICC and IF and has a HeLa IF image; A03770 also has a HeLa IF image but lists IF without ICC (catalog applications; A30486 and A03770 IF image captions). For Rat samples, A03770-1 is the only listed Rat-reactive option (catalog reactivity); its IHC image shows human brain tissue only, and the fixative is unreported in all three IHC image captions (each SKU’s IHC image caption).