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GSTA4 / Glutathione S-transferase A4 · IHC design guide
Design Immunohistochemistry for GSTA4
Plan chromogenic GSTA4 IHC in paraffin sections using the catalog antibody at 2–5 μg/ml (datasheet A03699-1). Compare cytoplasmic staining in Leydig and adrenal glandular cells, while interpreting signal cautiously because the tissue antibody may detect protein from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GSTA4 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in Leydig and adrenal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03699-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Appendix+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining may reflect protein from more than one gene (HPA tissue IHC) | |
| Regulation | No specific regulator reported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended GSTA4 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A03699-1). The published protocols below describe chromogenic GSTA4 IHC in mouse colon and human tissues (PMC10559223; PMC9619338).
| Sample | Paraffin-embedded human skeletal muscle tissue; fixative not specified (datasheet A03699-1) |
| Fixation | Image fixative and duration unreported (datasheet A03699-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03699-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03699-1) |
| Primary antibody | Rabbit anti-GSTA4, 2-5μg/ml (datasheet A03699-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03699-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03699-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GSTA4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, mainly in Leydig cells in testis, and glandular cells in adrenal gland and fallopian tube. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03699-1); consider glycine pH 9.0 for mouse colon (PMC10559223).
Section 2
What Is the Expected GSTA4 Staining Pattern?
GSTA4 is a cytoplasmic protein with no transmembrane segment (UniProt O15217). In paraffin-section IHC, look for cytoplasmic staining in Leydig cells of testis and glandular cells of adrenal gland; HPA also reports high staining in several other defined cell populations (HPA tissue IHC). Treat these as reference patterns rather than definitive specificity controls: HPA rates the tissue staining Approved, reports low agreement with RNA data, and cautions that the antibody targets protein from more than one gene (HPA tissue IHC).
| Cytoplasmic staining in adrenal glandular cells or testicular Leydig cells, with adjacent cells less prominent. | This matches the principal tissue profile and the High cell-level staining reported by HPA (HPA tissue IHC), as well as cytoplasmic localisation (UniProt O15217). Score the named cells and their compartment separately from whole-section colour; HPA’s Approved rating carries a low RNA–staining consistency caveat (HPA tissue IHC). |
| Predominantly nuclear, membrane-rim, or extracellular staining, without a convincing cytoplasmic component. | The compartment conflicts with the annotated cytoplasmic location and lack of a transmembrane segment (UniProt O15217). Suspect staining artefact or nonspecific detection first. This appearance alone cannot identify the cause; compare a known-positive cell population and the detection controls before calling it GSTA4. |
| Strong staining in an unexpected cell population, including one HPA lists as Not detected. | Unexpected distribution raises possible cross-reactivity or endogenous chromogen-generating activity, not proof of either. HPA specifically cautions that its tissue antibody targets protein from more than one gene (HPA tissue IHC). Interpret the named cell type: for example, HPA reports High in lung macrophages, while its fallopian-tube entries are internally inconsistent (HPA tissue IHC). |
| Diffuse colour across tissue, stroma, and slide background with little cell-specific contrast. | A uniform haze does not reproduce the cell-restricted HPA examples (HPA tissue IHC). In chromogenic IHC, excessive primary or detection reagent, inadequate blocking or washing, and endogenous enzyme activity are general possible causes (general IHC practice). Review the no-primary control and whether the expected cytoplasmic cells remain distinguishable. |
| No staining in the expected cells of an adrenal-gland or testis reference section. | An absent signal disagrees with HPA’s High glandular-cell or Leydig-cell observations (HPA tissue IHC), but a negative run does not establish absent GSTA4. Check that those cells are present and assess the IHC-validated antibody, retrieval, detection, and positive-control performance using general IHC practice. |
💡Expected GSTA4 appearanceCall a plausible positive when distinct cytoplasmic colour marks the expected cells, especially HPA High adrenal glandular cells or testicular Leydig cells (HPA tissue IHC; UniProt O15217); dominant nuclear or widespread cell-independent colour is suspect.
| Compartment and protein architecture (UniProt O15217). | GSTA4 is cytoplasmic, lacks a transmembrane segment, and has one annotated chain spanning residues 1–222 (UniProt O15217). These facts support a cytoplasmic reading of positive cells; they do not specify an antibody epitope or predict which retrieval condition will work. |
| Cell-level reference distribution (HPA tissue IHC). | HPA reports High staining in adrenal glandular cells, testicular Leydig cells, breast myoepithelial cells, duodenal endocrine cells, hepatocytes, and lung macrophages (HPA tissue IHC). The cell identity matters: HPA also lists Not detected populations in other tissues, so whole-organ labels alone are weak comparators. |
| Reference-pattern limitations (HPA tissue IHC). | HPA calls the tissue result Approved but flags low agreement between staining and RNA expression and protein targeting from more than one gene (HPA tissue IHC). Its profile mentions fallopian-tube glandular staining while its cell-level entry says Not detected; avoid treating that tissue as an unambiguous positive control. |
| Isoforms and assay interpretation (UniProt O15217; HPA antibodies). | UniProt lists 2 isoforms (UniProt O15217). The supplied antibody record gives IHC status as Approved and no epitope or isoform coverage (HPA antibodies). Thus, a tissue staining result cannot establish which isoform was detected or whether both are recognized. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reference adrenal gland or testis shows no expected cytoplasmic cells stained. | The selected field may lack the named cells, or the IHC run may have failed; HPA reports them as High reference populations (HPA tissue IHC). | Verify cell identity on the section, then check the run’s positive control, retrieval, primary-antibody use, and detection reagents (general IHC practice). Avoid interpreting an empty or unsuitable field as a biological negative. |
| Colour concentrates in nuclei or outlines cell membranes. | That distribution conflicts with GSTA4’s cytoplasmic annotation and absence of a transmembrane segment (UniProt O15217); nonspecific or detection-related staining is possible. | Compare the expected cytoplasm in HPA-positive cell populations, examine a no-primary control, and review staining conditions (HPA tissue IHC; general IHC practice). Do not score compartment-mismatched colour as a clean positive. |
| Many unrelated cells or extracellular areas carry similar chromogen. | Diffuse background or endogenous enzyme activity can obscure cell-specific signal in chromogenic IHC (general IHC practice). The HPA examples identify particular cell populations (HPA tissue IHC). | Inspect the no-primary control; review blocking, washing, reagent concentration, and endogenous-enzyme quenching as appropriate to the detection system (general IHC practice). Reassess only after the expected cells stand out from background. |
| Lung macrophages stain strongly, but surrounding respiratory cells differ. | HPA reports High staining in lung macrophages and Medium staining in bronchial respiratory epithelial cells (HPA tissue IHC); these are distinct tissue and cell entries. | Record the compartment and identity of each stained cell population. Compare like with like in the HPA tissue entries, and avoid treating every lung or airway cell as an equally strong control (HPA tissue IHC). |
| Fallopian-tube staining appears inconsistent with the HPA overview. | The HPA profile names fallopian-tube glandular cells, yet its detailed negative entry says those cells are Not detected (HPA tissue IHC). The supplied evidence does not resolve the discrepancy. | Document the observed cell type and cytoplasmic pattern, then use a less ambiguous HPA-positive reference such as adrenal glandular cells or testicular Leydig cells for run interpretation (HPA tissue IHC). |
| Can an IF/ICC image be used to confirm the IHC pattern? | HPA supplies no main ICC-IF location and no cell-line ICC-IF images for this record (HPA subcellular). Its tissue observations come from IHC (HPA tissue IHC). | Use the UniProt cytoplasmic annotation as an IF localisation expectation (UniProt O15217), while treating IF/ICC validation and controls as a separate guide question. Do not claim that HPA independently confirmed this IF pattern. |
Experiment evidence
IHC & IF images showing GSTA4 staining
A03699-1 · IHCIHC analysis of GSTA4 using anti-GSTA4 antibody (A03699-1). GSTA4 was detected in paraffin-embedded section of human skeletal muscle tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170899-GSTA4/tissue/cerebral+cortex#imid_14673483
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170899-GSTA4/tissue/colon#imid_14669830
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170899-GSTA4/tissue/liver#imid_14669815
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000170899-GSTA4/tissue/kidney#imid_14669816
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GSTA4 IHC & IF
🧪Run lung first and score cytoplasmic staining in macrophages (HPA: High in lung macrophages; UniProt O15217: cytoplasm). Run appendix endocrine cells as the negative tissue (HPA: Not detected in appendix endocrine cells). On the positive slide, cells lacking detectable GSTA4 should show only background staining; do not assume all neighboring cells are negative (HPA: High is specified for macrophages).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for GSTA4; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary and secondary-only controls, a matched nonimmune rabbit IgG isotype control for the catalog antibody, and GSTA4-knockout material as a biological negative (caption: rabbit anti-GSTA4; standard IHC practice). Block endogenous peroxidase and check endogenous biotin before SABC/DAB detection in lung tissue; assess macrophage autofluorescence if using IF (caption: SABC/DAB; standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03699-1 paraffin-section caption does not state the fixative (caption: fixative not stated). Its demonstrated IHC workflow uses heat retrieval in EDTA at pH 8.0, followed by 2 µg/mL primary antibody overnight at 4°C; whether retrieval is strictly required is unreported (caption: skeletal muscle IHC). Frozen sections and IF cannot be judged easier from the supplied evidence; lung macrophage autofluorescence may complicate IF interpretation (HPA: High in lung macrophages; standard IF practice).
HPA tissue IHC evidence for GSTA4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Colon | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GSTA4 IHC Tips
Troubleshoot GSTA4 staining in paraffin sections by checking retrieval, cellular localisation, antibody specificity, and cell resolved scoring before interpreting chromogenic signal.
What retrieval conditions should I start with for GSTA4 paraffin section IHC?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet A03699-1). The selected skeletal muscle image used this retrieval before incubation with 2 μg/ml catalog antibody overnight at 4°C (datasheet A03699-1). If staining is weak, compare a second retrieval condition on adjacent sections while holding antibody concentration, detection, and development constant (standard IHC practice). Include a section processed without primary antibody to distinguish retrieval related background from specific staining (standard IHC practice). Judge improvement by cytoplasmic signal in intact cells rather than stronger staining at tissue edges (UniProt O15217 localisation; standard IHC practice).
How should I troubleshoot weak GSTA4 staining when fixation history is uncertain?
The selected image identifies a paraffin embedded skeletal muscle section but does not state its fixative (datasheet A03699-1). Consequently, target specific fixation sensitivity is unknown; neither tissue staining patterns nor protein features establish how fixation changes this epitope (datasheet A03699-1; UniProt O15217; HPA tissue IHC). Record fixative, fixation duration, processing history, and section age for each specimen before comparing staining intensity (standard IHC practice). Compare specimens with matched processing and the same EDTA pH 8.0 retrieval and detection conditions (datasheet A03699-1; standard IHC practice). Treat a weak result from an undocumented specimen as inconclusive until a documented positive control stains appropriately (standard IHC practice).
What staining pattern is consistent with GSTA4 in tissue sections?
Evaluate GSTA4 primarily as cytoplasmic staining because the protein is assigned to the cytoplasm and has no transmembrane segment (UniProt O15217 localisation and topology). The selected skeletal muscle paraffin section demonstrates tissue IHC staining with the catalog antibody, although its caption does not specify a subcellular pattern (datasheet A03699-1). Compare intact cell bodies with nuclei and tissue boundaries under the same counterstain and illumination conditions (standard IHC practice). A predominantly nuclear or sharply membranous pattern warrants review of controls and morphology before attribution to GSTA4 (UniProt O15217 localisation; standard IHC practice). Record the compartment separately from intensity so an unusual pattern remains visible in the results (standard IHC practice).
Can this stain distinguish GSTA4 isoforms or confirm its antibody epitope?
GSTA4 has 2 reported isoforms, but the supplied caption does not define which sequence the antibody recognises (UniProt O15217 isoforms; datasheet A03699-1). The protein contains GST N terminal residues 3–83 and GST C terminal residues 85–208; those domain boundaries do not locate this antibody's epitope (UniProt O15217 domains; datasheet A03699-1). Do not assign an isoform from chromogenic staining intensity or cytoplasmic localisation alone (UniProt O15217 isoforms and localisation; standard IHC practice). Seek an epitope map or isoform specific validation before making that claim (standard IHC practice). HPA flags possible recognition of proteins from more than one gene, so tissue staining alone cannot establish molecular specificity (HPA tissue IHC reliability description).
How should I assess GSTA4 by IF alongside this chromogenic IHC assay?
Treat IF as a separate assay requiring its own antibody and imaging validation; the selected evidence describes paraffin section chromogenic IHC (datasheet A03699-1). Multiplex GSTA4 with a validated marker for the expected cell population, such as macrophages in lung, and confirm both signals within individual cells (HPA: High in lung macrophages; standard IF practice). Choose fluorophores after checking unstained tissue autofluorescence in each channel, then include single colour controls for bleed through (standard IF practice). Because GSTA4 is cytoplasmic with no transmembrane segment, permeabilisation must allow antibody access to the intracellular epitope (UniProt O15217 localisation and topology; standard IF practice). Do not transfer the IHC retrieval conditions into an IF protocol without testing (datasheet A03699-1; standard IF practice).
How can I separate GSTA4 signal from chromogenic background?
The selected IHC image used 10% goat serum block, a biotinylated secondary antibody, a streptavidin biotin complex, and DAB (datasheet A03699-1). Run no primary and secondary only controls to assess detection background, and apply a peroxidase block as a general DAB workflow step (standard IHC practice). If diffuse colour persists, inspect endogenous enzyme activity, biotin dependent signal, and excessive chromogen development before changing the primary antibody concentration (standard IHC practice). Compare background in unstained structures with cytoplasmic signal in morphologically intact cells, using identical exposure and development across sections (UniProt O15217 localisation; standard IHC practice). Note tissue edges and damaged regions separately because their staining may be misleading (standard IHC practice).
How should I score GSTA4 staining across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, consistent with GSTA4 localisation (UniProt O15217 localisation; standard IHC practice). For each population, record percent positive cells and intensity categories, then calculate an H score only when those categories are consistently applied (standard IHC practice). Normalise positive counts to all eligible cells of that population, or report density per mm² of viable tissue when cell counting is impractical (standard IHC practice). Keep section thickness, retrieval, DAB development, and image acquisition consistent across compared samples (standard IHC practice). Report cell type separately because HPA lists High staining in lung macrophages and liver hepatocytes, which represent different denominators (HPA tissue IHC).
When should I question an apparent GSTA4 positive result?
Expect cytoplasmic signal in intact cells; dominant nuclear or crisp membranous staining conflicts with the reported localisation and topology (UniProt O15217 localisation and topology). Check whether the stained cell type matches the claimed result: HPA reports High staining in testicular Leydig cells and lung macrophages, but its reliability note warns of low agreement with RNA and possible recognition of multiple genes (HPA tissue IHC). Exclude tissue edges, necrotic areas, and diffuse deposits when judging a positive cell (standard IHC practice). A signal reproduced in the no primary control may reflect endogenous enzyme or detection background rather than antibody binding (standard IHC practice). Interpret a convincing pattern alongside morphology, controls, and independent evidence of expression (standard IHC practice).
Boster reagents
Best GSTA4 / Glutathione S-transferase A4 IHC Antibodies
The catalog antibody has real IHC data from paraffin-embedded human skeletal muscle (IHC figure caption). Human and monkey reactivity are listed; IF/ICC validation is absent (catalog applications and images).
Real IHC data
Anti-GSTA4 Antibody ®
Cat # A03699-1
A03699-1 is listed for IHC and has a figure showing chromogenic detection in a paraffin-embedded human skeletal muscle section (catalog applications; IHC figure caption). Its listed reactivity is human and monkey, with no IF/ICC application or image supplied (catalog reactivity, applications and images).
Which to pick: Choose A03699-1 for paraffin-section tissue IHC: its own figure shows staining of human skeletal muscle, and the catalog specifies 2–5 μg/ml for IHC (IHC figure caption; datasheet: IHC dilution). The caption does not report the fixative (IHC figure caption). No SKU in this payload has IF/ICC validation; A03699-1 lists monkey reactivity, but its IHC figure documents human tissue only (catalog applications, images and reactivity; IHC figure caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15217 (GSTA4_HUMAN, Glutathione S-transferase A4).
- Human Protein Atlas. GSTA4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. GSTA4 subcellular location (ICC-IF): Highest expression in AF22: 89.2 nTPM.
- Human Protein Atlas. GSTA4 antibody validation summary (1 antibodies).
- Colonic expression of glutathione S-transferase alpha 4 and 4-hydroxynonenal adducts is correlated with the pathology of murine colitis-associated cancer. Heliyon 2023 — PMC10559223.
- Glutathione S-transferase alpha 4 induction by activator protein 1 in colorectal cancer. Oncogene 2016 — PMC9619338.
- Nuclear receptors regulate lipid metabolism and oxidative stress markers in chondrocytes. Journal of molecular medicine (Berlin, Germany) 2017 — PMC5357281.
- GSTA4 mediates reduction of cisplatin ototoxicity in female mice. Nature communications 2019 — PMC6742643.
- PubMed PMID:9461507 — UniProt-cited evidence.
- PubMed PMID:9480897 — UniProt-cited evidence.
- PubMed PMID:9587421 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GSTA4 (Glutathione S-transferase A4). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03699-1). Validated IHC antibody: A03699-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Appendix. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human skeletal muscle tissue; fixative not specified (datasheet A03699-1); 2. Fixation: Image fixative and duration unreported (datasheet A03699-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A03699-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03699-1); 8. Primary antibody: Rabbit anti-GSTA4, 2-5μg/ml (datasheet A03699-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03699-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining may reflect protein from more than one gene (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences are unspecified (UniProt); Regulation: No specific regulator reported (UniProt). Evidence: UniProt O15217, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in AF22: 89.2 nTPM, Datasheet A03699-1, PMC10559223, PMC9619338. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GSTA4/immunohistochemistry-ihc-guide.