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GSPT1 / Eukaryotic peptide chain release factor GTP-binding subunit ERF3A · IHC design guide
Design Immunohistochemistry for GSPT1
Plan chromogenic GSPT1 IHC on paraffin sections with the IHC-validated antibody at 1:100–1:300 (datasheet: A07761). Expect broad cytoplasmic staining, with high staining in several glandular cell populations and no detected staining in adipocytes (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GSPT1 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 staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Broad cytoplasmic staining, often high in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep tissue fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Adipocytes may be unstained despite broad tissue expression (HPA tissue IHC) | |
| Regulation | Expression regulation is not documented (UniProt) | |
| Isoform / epitope | 3 isoforms; antibody epitope coverage is unspecified (UniProt; datasheet) |
Section 1
Recommended GSPT1 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol with four published GSPT1 IHC methods (PMC9640916; PMC9937881; PMC8064227; PMC8798158).
| Sample | Paraffin-embedded human prostate carcinoma tissue; fixative not specified (datasheet A07761) |
| Fixation | Image fixative and duration unreported (datasheet A07761); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-GSPT1, 1:100-1:300 (datasheet A07761) |
| 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 | GSPT1-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval). Use an article’s stated retrieval conditions when reproducing its method (PMC8064227; PMC8798158).
Section 2
What Is the Expected GSPT1 Staining Pattern?
GSPT1 should show cytoplasmic staining across many cell types, with high signal in several glandular and epithelial populations and bone marrow hematopoietic cells (HPA tissue IHC: ubiquitous cytoplasmic expression; listed high staining). Its lack of a transmembrane segment supports a nonmembranous pattern (UniProt P15170 topology). HPA rates the tissue IHC profile Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in glandular, respiratory epithelial, or hematopoietic cells. | This fits the reported pattern. HPA lists high staining in appendix, breast, colon, duodenum, endometrium, and epididymis glandular cells, bronchial respiratory epithelial cells, and bone marrow hematopoietic cells (HPA tissue IHC). Compare intensity within the cell type being assessed; a high result in one listed tissue does not set a universal threshold. |
| Predominantly nuclear staining, or a crisp membrane outline with little cytoplasmic signal. | Treat this as discordant with the reported cytoplasmic IHC pattern (HPA tissue IHC) and cytosolic ICC-IF location (HPA subcellular). A membrane outline also lacks support from the sequence topology (UniProt P15170 topology). Review the negative control and detection conditions before assigning the signal to GSPT1. |
| Strong staining in adipocytes while nearby expected positive cells stain weakly. | HPA reports adipocytes in adipose tissue as not detected (HPA tissue IHC). Consider cross-reactivity or endogenous chromogen activity, particularly if the negative control also develops color; those are possible IHC artifacts, not established explanations for this specimen. Recheck cell identification and controls. |
| Diffuse color over stroma, empty spaces, or most cells without clear cytoplasmic boundaries. | This distribution does not resolve the reported cellular pattern (HPA tissue IHC: ubiquitous cytoplasmic expression). General IHC causes include nonspecific binding, incomplete blocking, or excess detection background. Interpret intensity only after background is low enough to distinguish stained cytoplasm from the surrounding section. |
| No staining in a section containing a listed high-staining cell population. | A failed positive control or insufficient assay sensitivity is possible when glandular, respiratory epithelial, or hematopoietic cells expected to stain high remain blank (HPA tissue IHC: listed high staining). Check the complete staining run before calling GSPT1 absent; HPA's medium RNA–protein consistency limits certainty for any individual specimen (HPA tissue IHC). |
💡Expected GSPT1 appearanceCall a result positive when identifiable cells show cytoplasmic chromogen, with stronger signal expected in HPA-listed high-staining populations; isolated nuclear, membrane-rim, or control-matched diffuse color is suspect (HPA tissue IHC; HPA subcellular; UniProt P15170 topology).
| Tissue and cell selection | HPA reports low tissue specificity overall, yet staining varies by cell population: listed glandular, bronchial epithelial, and marrow hematopoietic cells are high; adipocytes are not detected (HPA tissue IHC). Select a control by the reported cell type, not by an assumption that every cell in a positive organ stains equally. |
| Strength of IHC evidence | The tissue profile is Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC). HPA052488 is IHC Approved, whereas the supplied record gives no IHC status for HPA074588 (HPA antibodies). These labels support a reference pattern, not proof that every positive signal is specific. |
| Topology and processing | The protein has no transmembrane segment, signal peptide, or propeptide, and its recorded chain spans residues 1–499 (UniProt P15170 topology and processing). A sharp surface-only pattern therefore needs independent support. The record does not identify an IHC epitope or establish how retrieval affects it. |
| Isoform coverage | UniProt lists 3 isoforms (UniProt P15170 isoforms). The supplied evidence does not map the IHC antibody epitope across them, so staining cannot be assigned to a particular isoform. Check documented epitope coverage if an isoform-specific interpretation is required. |
| IF/ICC: what localization should I expect? | Mainly cytosolic signal, with additional vesicular localization, is the approved ICC-IF observation (HPA subcellular). This is a separate cellular readout; the paraffin IHC reference is ubiquitous cytoplasmic staining (HPA tissue IHC). The supplied evidence provides no IF/ICC protocol to recommend. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known positive cell population is blank. | The run may lack detectable signal; HPA lists high staining in several glandular, bronchial epithelial, and marrow hematopoietic populations (HPA tissue IHC). | Inspect the positive control and reagent steps, then review retrieval, antibody incubation, and chromogen development as general IHC workflow checks. Do not infer a GSPT1-specific fixation effect; none is supplied. |
| Nuclear or membrane-rim color dominates. | The pattern conflicts with HPA cytoplasmic IHC and approved cytosolic ICC-IF localization (HPA tissue IHC; HPA subcellular). GSPT1 has no annotated transmembrane segment (UniProt P15170 topology). | Compare the same compartment in a listed high-staining control and its negative control. Review cell boundaries and detection background before scoring the unusual compartment as target signal. |
| Adipocytes show strong color. | Adipocytes are reported not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible under general IHC practice. | Confirm the stained cells are adipocytes, inspect the negative control for matching color, and reassess blocking and detection chemistry. Keep the result provisional until the discordance is resolved. |
| The entire section looks hazy or uniformly brown. | Diffuse background can obscure the expected cytoplasmic distribution (HPA tissue IHC: ubiquitous cytoplasmic expression). Excess reagent or inadequate washing are general IHC possibilities. | Compare background with the negative control, then review antibody concentration, wash steps, blocking, and chromogen time as general workflow variables. Score cells only where their cytoplasm remains distinguishable. |
| One listed positive tissue stains much less than another. | HPA reports high staining for specific cell populations, but its tissue IHC profile has only medium consistency with RNA expression (HPA tissue IHC). Different cell composition can also change the apparent field intensity. | Compare like cell types at the same scoring threshold and include a high-staining reference population from the same run (HPA tissue IHC). Confirm cellular localization before interpreting a weak field as absent expression. |
Experiment evidence
IHC & IF images showing GSPT1 staining
A07761 · IHCImmunohistochemistry analysis of paraffin-embedded human prostate carcinoma tissue, using GSPT1 Antibody. The picture on the right is blocke
A07761-1 · IHCImmunohistochemistry (IHC) analyzes of eRF3a (D128) pAb in paraffin-embedded human colorectal carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000103342-GSPT1/tissue/cerebral+cortex#imid_15069798
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000103342-GSPT1/tissue/colon#imid_15069716
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000103342-GSPT1/tissue/liver#imid_15069717
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000103342-GSPT1/tissue/pancreas#imid_15069668
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA052488; green = GSPT1, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA052488; green = GSPT1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GSPT1 IHC & IF
🧪Run appendix first: its glandular cells should stain strongly (HPA: High in appendix glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); no internal negative cell population is established for the appendix slide, so any unstained neighboring cells serve only as background references.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GSPT1 in HeLa, MCF-7, U2OS, PC-3, SiHa, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an irrelevant IgG control matched to the primary antibody’s host species and class, and a peptide-block control (selected-SKU tissue-IHC caption: staining blocked with synthesized peptide). For chromogenic detection, block endogenous peroxidase in appendix sections and check background in the no-primary control (standard IHC practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; the paraffin-section caption does not report a fixative (selected-SKU tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier than IHC-P, although ICC-IF images show mainly cytosolic signal (HPA subcellular: Cytosol approved). Assess appendix sections for endogenous peroxidase background before interpreting glandular staining (standard chromogenic IHC practice).
HPA tissue IHC evidence for GSPT1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GSPT1 IHC Tips
Troubleshoot chromogenic IHC for GSPT1 in paraffin sections using the catalog antibody, with a separate IF/ICC question for multiplex experiments.
What retrieval should I start with for weak GSPT1 staining?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Let sections cool in buffer, then compare staining with an otherwise identical section processed without retrieval to assess whether heating improves signal (standard IHC practice). GSPT1 has no transmembrane segment, and HPA reports mainly cytosolic staining, so assess signal within cells rather than at their borders (UniProt P15170 topology; HPA subcellular). If staining remains weak, adjust heating time in a small side-by-side series while keeping antibody and detection conditions fixed (standard IHC practice). Record tissue morphology alongside signal, because excessive heating can damage sections and complicate scoring (standard IHC practice).
Could fixation explain inconsistent GSPT1 staining across paraffin sections?
The A07761 image documents staining in paraffin-embedded human prostate carcinoma, but its caption does not state the fixative (A07761 tissue-IHC caption). Target-specific sensitivity to fixation is therefore unknown; neither HPA staining patterns nor GSPT1 topology or modification records establish it (A07761 tissue-IHC caption; HPA tissue IHC; UniProt P15170). For a controlled comparison, document fixation conditions and process matched sections with the same pH 6.0 citrate retrieval for 20 min (page retrieval setting; standard IHC practice). Compare staining intensity, cellular distribution and morphology across those sections before changing antibody incubation or detection (standard IHC practice). Include the same control material in each staining run to identify variation introduced during processing (standard IHC practice).
Should GSPT1 stain nuclei, cytoplasm, or discrete vesicles?
Expect predominantly cytoplasmic chromogenic staining: HPA reports ubiquitous cytoplasmic tissue expression and approves cytosol as the main subcellular location (HPA tissue IHC; HPA subcellular). HPA also reports vesicles as an additional location, so occasional intracellular puncta can be assessed alongside diffuse cytoplasmic signal (HPA subcellular). UniProt supplies no subcellular annotation and reports no transmembrane segment; neither record supports treating a crisp cell-border pattern as the expected finding (UniProt P15170). Examine both glandular and surrounding cells at the same magnification, using a 20 min citrate retrieval run as a consistent comparison condition (page retrieval setting; standard IHC practice). If staining appears mainly nuclear, repeat with a negative control and review counterstain overlap before assigning it to GSPT1 (standard IHC practice).
How can isoforms or epitope accessibility affect this IHC result?
GSPT1 has 3 listed isoforms, but the supplied A07761 caption does not identify the antibody epitope or establish which isoforms it recognizes (UniProt P15170; A07761 tissue-IHC caption). Consequently, a weak or absent stain cannot by itself establish loss of every GSPT1 isoform (UniProt P15170; standard IHC interpretation). The caption reports a synthesized-peptide blocking image in paraffin-embedded human prostate carcinoma; use that comparison as evidence for competition under its stated conditions (A07761 tissue-IHC caption). Keep retrieval at citrate pH 6.0 for 20 min when comparing sections, because changing retrieval can also change epitope accessibility (page retrieval setting; standard IHC practice). Confirm isoform claims only after obtaining epitope and isoform-recognition information for the antibody (standard antibody-validation practice).
How should I check GSPT1 localisation in a multiplex IF experiment?
For the separate IF/ICC workflow, choose a marker that identifies the cell population being examined and score GSPT1 within those marked cells (standard multiplex IF practice). HPA places GSPT1 mainly in the cytosol, with an additional vesicular location, so permeabilise fixed cells for access to intracellular epitopes and titrate that step against morphology (HPA subcellular; standard IF practice). Use a far-red GSPT1 fluorophore when tissue autofluorescence is prominent, and inspect single-stain and unstained controls before interpreting overlap (standard IF practice). Capture each channel with the same exposure settings across comparison groups, then assess cytosolic signal rather than inferring membrane staining from adjacent channels (standard IF practice; HPA subcellular). The 3 listed isoforms make epitope documentation relevant to any claim that IF detects all variants (UniProt P15170).
How do I distinguish GSPT1 staining from chromogenic background?
Check a section processed without primary antibody to reveal detection-system and tissue background, and inspect endogenous pigment before assigning brown signal to DAB (standard IHC practice). Apply a peroxidase block for a peroxidase-based detection workflow; this is a general IHC step, not evidence that GSPT1 itself causes background (standard IHC practice). HPA reports high GSPT1 staining in bone-marrow hematopoietic cells and several glandular cell populations, so staining in those cells is not automatically nonspecific (HPA tissue IHC). Compare cytoplasmic signal after the same citrate pH 6.0, 20 min retrieval with the negative control and with adjacent intact tissue (page retrieval setting; HPA tissue IHC; standard IHC practice). Exclude precipitate, folds and section edges from assessment (standard IHC practice).
What scoring method best captures GSPT1 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue compartment before scoring, then report the percentage of positive cells and cytoplasmic intensity using the same threshold across sections (standard IHC quantification; HPA tissue IHC). An H-score can combine percentages at intensity grades 0–3 into a 0–300 summary, provided the grading rules are fixed before comparison (standard IHC quantification). Normalise counts to the number of evaluable cells, or report positive-cell density per mm² of viable tissue when area is the denominator (standard IHC quantification). Exclude necrosis, folds and poorly preserved regions, and record which compartments were omitted (standard IHC quantification). Because HPA describes broad cytoplasmic expression, compare like cell types and staining runs rather than treating whole-section color as a cell-specific measure (HPA tissue IHC; standard IHC quantification).
When is a GSPT1-positive IHC result convincing rather than artefactual?
A convincing result shows reproducible intracellular, chiefly cytoplasmic staining in intact cells, consistent with HPA tissue and subcellular observations (HPA tissue IHC; HPA subcellular; standard IHC interpretation). HPA reports high staining in appendix glandular cells and bone-marrow hematopoietic cells, while adipocytes are listed as not detected; use the appropriate cell type when judging a field (HPA tissue IHC). Isolated nuclear or sharp membrane staining, staining restricted to section edges or necrosis, and signal in the no-primary control warrant investigation (HPA subcellular; standard IHC interpretation). The A07761 prostate-carcinoma caption includes a synthesized-peptide blocking image, which supports competition in that image but does not independently establish every stained cell as specific (A07761 tissue-IHC caption; standard IHC interpretation). Check endogenous peroxidase and compare matched pH 6.0, 20 min retrieval runs before interpreting a biological difference (standard IHC practice; page retrieval setting).
Boster reagents
Best GSPT1 / Eukaryotic peptide chain release factor GTP-binding subunit ERF3A IHC Antibodies
Both anti-GSPT1 antibodies have IHC images from paraffin-embedded human carcinoma tissue (catalog image captions) and list human, mouse, and rat reactivity (catalog reactivity); only A07761 lists IF (catalog applications).
Real IHC data
Anti-eRF3a GSPT1 Antibody
Cat # A07761
Real IHC data
Anti-eRF3a (D128) GSPT1 Antibody
Cat # A07761-1
A07761 will render with IHC of paraffin-embedded human prostate carcinoma and a peptide-blocked comparison (A07761 image caption); its application list includes IF (A07761 catalog applications). A07761-1 will render with IHC of paraffin-embedded human colorectal carcinoma at 1:50 (A07761-1 image caption).
Which to pick: For tissue IHC, choose either SKU according to the pictured sample: A07761 shows paraffin-embedded human prostate carcinoma, while A07761-1 shows paraffin-embedded human colorectal carcinoma (respective image captions); neither caption reports the fixative (respective image captions). For IF, choose rabbit polyclonal A07761 because IF is listed (A07761 catalog applications and dilution record); ICC validation and an IF image are unreported (A07761 catalog applications and image alts). For mouse or rat work, both list reactivity in those species (catalog reactivity), although their IHC images show human tissue only (respective image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P15170 (ERF3A_HUMAN, Eukaryotic peptide chain release factor GTP-binding subunit ERF3A).
- Human Protein Atlas. GSPT1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. GSPT1 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to vesicles..
- Human Protein Atlas. GSPT1 antibody validation summary (2 antibodies).
- An integrative analysis of the lncRNA-miRNA-mRNA competitive endogenous RNA network reveals potential mechanisms in the murine hair follicle cycle. Frontiers in genetics 2022 — PMC9640916.
- Hsa_circ_0001944 enhanced GSPT1 expression via sponging miR-498 to promote proliferation and invasion of gastric cancer. Journal of clinical laboratory analysis 2023 — PMC9937881.
- Identification of GSPT1 as prognostic biomarker and promoter of malignant colon cancer cell phenotypes via the GSK-3β/CyclinD1 pathway. Aging 2021 — PMC8064227.
- Long non-coding RNA LINC00511 promotes proliferation, invasion, and migration of non-small cell lung cancer cells by targeting miR-625-5p/GSPT1. Translational cancer research 2021 — PMC8798158.
- PubMed PMID:2511002 — UniProt-cited evidence.
- PubMed PMID:10493829 — UniProt-cited evidence.
- PubMed PMID:15616553 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GSPT1 (Eukaryotic peptide chain release factor GTP-binding subunit ERF3A). Expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A07761 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate carcinoma tissue; fixative not specified (datasheet A07761); 2. Fixation: Image fixative and duration unreported (datasheet A07761); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-GSPT1, 1:100-1:300 (datasheet A07761); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep tissue fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Adipocytes may be unstained despite broad tissue expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; antibody epitope coverage is unspecified (UniProt; datasheet); Regulation: Expression regulation is not documented (UniProt). Evidence: UniProt P15170, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to vesicles., Datasheet A07761, PMC9640916, PMC9937881. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GSPT1/immunohistochemistry-ihc-guide.