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EIF6 / Eukaryotic translation initiation factor 6 · IHC design guide
Design Immunohistochemistry for EIF6
Plan EIF6 paraffin IHC using tissue controls and score cytoplasmic and nuclear staining (HPA tissue IHC). Colon carcinoma has higher EIF6 protein expression than normal colon (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EIF6 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 and nuclear in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9582) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+2 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). Selected-image fixative and duration unreported (datasheet PB9582); verify before use. | |
| Caveat | Adipocytes and myocytes have no detected staining (HPA tissue IHC) | |
| Regulation | Colon carcinoma exceeds normal colon (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended EIF6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published EIF6 staining methods for intestinal-type adenocarcinoma and hypertrophic scar (PMC8616251; PMC4736889).
| Sample | Paraffin-embedded Mouse Intestine tissue; fixative not specified (datasheet PB9582) |
| Fixation | Image fixative and duration unreported (datasheet PB9582); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9582) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9582) |
| Primary antibody | Rabbit anti-EIF6, 0.5-1μg/ml (datasheet PB9582) |
| Primary incubation | Overnight at 4 °C (datasheet PB9582) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9582) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EIF6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet PB9582); the published methods use mCC1/sCC1 (PMC8616251) or Proteinase K (PMC4736889).
Section 2
What Is the Expected EIF6 Staining Pattern?
EIF6 is expected in the cytoplasm and nucleus of many tissue cells, with nucleolar localisation also reported (UniProt P56537). HPA describes cytoplasmic and nuclear staining in most tissues and rates its tissue IHC evidence Enhanced (HPA: tissue IHC). EIF6 has no transmembrane segment, so a membrane-restricted pattern is unexpected (UniProt P56537 topology).
| Cytoplasmic and nuclear staining in glandular epithelium, including colon or appendix (HPA: High in glandular cells). | This matches the broad tissue pattern reported by HPA (HPA: tissue IHC). Score each compartment and cell population separately; a mixed pattern is consistent with EIF6 shuttling between cytoplasm and nucleus (UniProt P56537). |
| Crisp membrane-only or extracellular staining, with little intracellular signal. | This conflicts with the reported cytoplasmic and nuclear localisation (HPA: tissue IHC; UniProt P56537). EIF6 has no transmembrane segment (UniProt P56537 topology); check whether the pattern persists in control sections before assigning it to EIF6. |
| Strong staining of adipocytes or skeletal or smooth muscle cells (HPA: Not detected in those cells). | That distribution conflicts with the listed HPA IHC observations and warrants a specificity check (HPA: tissue IHC). Cross-reactivity or endogenous detection activity are possible explanations (general IHC practice), not established properties of the antibody. |
| Uniform chromogen across cells and tissue spaces, obscuring cell boundaries. | A diffuse deposit cannot establish the reported intracellular EIF6 pattern (HPA: tissue IHC). Review the no-primary control for background and endogenous detection activity, and compare it with the test section under the same imaging conditions (general IHC practice). |
| No signal in colon glandular cells or kidney tubule cells (HPA: High in both populations). | A negative result there needs a technical check before biological interpretation (HPA: tissue IHC). Confirm tissue identity and morphology, then review the IHC-validated antibody, detection reagents and the run’s positive control (general IHC practice). |
💡Expected EIF6 appearanceCall a section positive when identifiable cells show cytoplasmic and nuclear staining, such as high staining in colon glandular cells (HPA: tissue IHC); membrane-only staining is suspect given EIF6 localisation and topology (UniProt P56537).
| Compartment and topology | EIF6 shuttles between cytoplasm and nucleus/nucleolus and has no transmembrane segment (UniProt P56537). Evaluate intracellular compartments rather than treating a membrane rim as the expected result. |
| Choice of comparison tissue | HPA reports High staining in colon glandular cells and kidney tubule cells, but Not detected in adipocytes and skeletal or smooth muscle cells (HPA: tissue IHC). These are cell-level comparisons, not whole-organ positive or negative labels. |
| Strength of IHC evidence | HPA rates its tissue IHC evidence Enhanced, citing consistency between antibody staining and RNA expression; HPA040873 is also listed as IHC Enhanced (HPA: tissue IHC; HPA: antibody validation). These ratings support interpretation but do not validate every staining condition. |
| Molecular annotations | UniProt lists two isoforms, one chain spanning residues 1–245, and modified residues (UniProt P56537). Without an epitope map or target-specific retrieval evidence, these annotations do not predict which isoform a stain detects or how retrieval changes signal. |
| IF/ICC Q: Should its image match IHC exactly? | A: HPA supports nucleoplasmic localisation in ICC-IF, while its tissue IHC profile is cytoplasmic and nuclear (HPA: subcellular; HPA: tissue IHC). Interpret each application against its own evidence; this guide gives no IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive glandular population appears blank. | The result conflicts with High staining reported for colon glandular cells (HPA: tissue IHC); the cause is undetermined. | Check the section’s morphology and cell identity, then verify the IHC-validated antibody and detection run with a positive control (general IHC practice). |
| Only a sharp membrane rim is visible. | The pattern conflicts with intracellular localisation and absence of a transmembrane segment (HPA: tissue IHC; UniProt P56537). | Compare with a no-primary control and an expected positive cell population before scoring the rim as EIF6 (general IHC practice). |
| Adipocytes or muscle cells stain strongly. | HPA lists EIF6 as Not detected in those cell types (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Check cell identity and the no-primary control; assess whether expected glandular or epithelial cells show the reported pattern (HPA: tissue IHC; general IHC practice). |
| The entire section has a diffuse deposit. | Nonspecific background or endogenous detection activity can obscure localisation (general IHC practice). | Review the no-primary control, blocking and detection steps; interpret EIF6 only where cell boundaries and intracellular staining remain distinguishable (general IHC practice). |
| Nuclear staining dominates an IHC section. | Nuclear EIF6 is plausible, but HPA’s tissue IHC profile also includes cytoplasmic expression (HPA: tissue IHC; UniProt P56537). | Score nuclear and cytoplasmic signal separately across identifiable cells; compare with HPA’s IHC pattern without requiring every cell to have identical balance (HPA: tissue IHC). |
| ICC-IF appears nucleoplasmic while tissue IHC has cytoplasmic signal. | The application-specific observations differ: HPA supports nucleoplasm in ICC-IF and reports cytoplasmic and nuclear tissue IHC (HPA: subcellular; HPA: tissue IHC). | Use the relevant application’s localisation evidence when interpreting each image; do not treat this difference alone as assay failure (HPA: subcellular; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing EIF6 staining
PB9582 · IHCIHC analysis of EIF6 using anti-EIF6 antibody (PB9582). EIF6 was detected in paraffin-embedded section of Mouse Intestine Tissue. Heat media
PB9582 · IHCIHC analysis of EIF6 using anti-EIF6 antibody (PB9582). EIF6 was detected in paraffin-embedded section of Rat Testis Tissue. Heat mediated a
M03581 · IHCIHC analysis of EIF6 using anti-EIF6 antibody (M03581). EIF6 was detected in paraffin-embedded section of human intestinal cancer tissue. He
M03581 · IHCIHC analysis of EIF6 using anti-EIF6 antibody (M03581). EIF6 was detected in paraffin-embedded section of human mammary cancer tissue. Heat
PA2031 · IHCAnti-EIF6 antibody, PA2031, IHC(P) IHC(P): Human Intestinal Cancer Tissue
PA2031 · IHCAnti-EIF6 antibody, PA2031, IHC(F) IHC(F): Human Placenta Tissue
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000242372-EIF6/tissue/cerebral+cortex#imid_11210805
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000242372-EIF6/tissue/esophagus#imid_11210763
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000242372-EIF6/tissue/colon#imid_11210826
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000242372-EIF6/tissue/liver#imid_11210831
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040873; green = EIF6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040873; green = EIF6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EIF6 IHC & IF
🧪Run colon first: its glandular cells should stain (HPA: High in colon glandular cells). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); assess background in unstained cells on the colon slide, without treating a specific internal cell type as a proven EIF6-negative control.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EIF6 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a species- and clonality-matched rabbit isotype control for PB9582, and an EIF6 knockout sample or validated immunizing-peptide block as a biological specificity control (caption: rabbit anti-EIF6 PB9582). For the SABC/DAB workflow, block endogenous peroxidase and check for endogenous biotin background in the positive tissue (caption: SABC/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (caption: fixative not stated). The reported IHC workflow uses heat retrieval in citrate buffer at pH 6 for 20 minutes; retrieval dependence beyond that workflow is unreported (caption: citrate retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; ICC-IF images support nucleoplasmic localisation, while SABC/DAB detection calls for a background check in the colon control (HPA: Nucleoplasm supported; caption: SABC/DAB detection).
HPA tissue IHC evidence for EIF6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Section 3
Advanced EIF6 IHC Tips
Troubleshoot EIF6 staining in paraffin sections by checking retrieval, compartment pattern, controls and cell-specific scoring before interpreting intensity.
How should I optimize retrieval when EIF6 staining is weak in paraffin sections?
Start with heat-mediated citrate buffer at pH 6 for EIF6 paraffin-section IHC (datasheet PB9582). The selected mouse intestine image used 20 minutes of retrieval, followed by 1 μg/ml primary antibody overnight at 4°C (PB9582 tissue-IHC caption). If signal is weak, compare retrieval durations on adjacent sections while holding antibody concentration, detection and imaging conditions constant (standard IHC practice). Include a known positive compartment in each run: EIF6 can appear in cytoplasm and nucleus, including the nucleolus, so a purely negative section does not isolate retrieval failure (UniProt P56537 subcellular location).
Could fixation explain weak or uneven EIF6 staining?
EIF6-specific fixation sensitivity is unknown from the supplied evidence; the selected paraffin-section caption does not state its fixative (PB9582 tissue-IHC caption). Record the fixative, fixation duration, tissue thickness and processing history before comparing staining across blocks (standard IHC practice). If staining varies, process matched sections with the same citrate pH 6 retrieval and detection conditions, then compare preserved morphology alongside signal (datasheet PB9582; standard IHC practice). Avoid attributing a weak result to EIF6 phosphorylation or cellular location alone: these are documented protein features, but neither establishes a fixation response (UniProt P56537 modified residues and subcellular location).
Should I expect nuclear, nucleolar or cytoplasmic EIF6 staining?
Score nuclear and cytoplasmic signal separately because EIF6 shuttles between cytoplasm and nucleus or nucleolus (UniProt P56537 subcellular location). Broad cytoplasmic and nuclear tissue staining is reported, while the separate cell-based localisation summary supports nucleoplasmic staining (HPA tissue IHC profile; HPA subcellular summary). Examine intact cells at high magnification and compare each compartment with its immediate background before calling a faint signal positive (standard IHC practice). If one preparation shows only a single compartment, check retrieval, counterstain intensity and focal plane, then repeat with the same settings; localisation differences alone do not establish altered EIF6 abundance (standard IHC practice; UniProt P56537 subcellular location).
Can this staining distinguish EIF6 isoforms or phosphorylation states?
Do not assign this catalog antibody’s staining to a particular isoform or phospho-state without epitope and specificity evidence for that distinction (standard IHC practice). EIF6 has 2 annotated isoforms and several modified residues, including sites at 113, 165, 166, 174 and 175 (UniProt P56537 isoforms and modified residues). Its annotated chain spans residues 1–245, with no signal peptide or propeptide, but these annotations do not identify the antibody’s epitope (UniProt P56537 processing). When a condition changes staining, keep retrieval and exposure matched, inspect subcellular distribution, and seek a separately validated isoform- or modification-specific reagent before making a molecular assignment (standard IHC practice).
How can I investigate an EIF6 IHC pattern with multiplex immunofluorescence?
Use IF as a separate validation experiment and pair EIF6 with a validated marker for the cell population whose IHC signal you are testing (standard IF practice). For glandular cells in colon, that pairing can clarify whether signal follows the expected population; HPA reports high EIF6 staining in those cells (HPA tissue IHC: High in colon glandular cells). Choose spectrally separated fluorophores after checking tissue autofluorescence, and include single-stain and no-primary controls when assessing overlap (standard IF practice). EIF6 has no transmembrane segment and occurs in cytoplasm and nucleus or nucleolus, so optimize permeabilisation for intracellular access while checking morphology; the IHC caption does not establish IF conditions (UniProt P56537 topology and subcellular location; PB9582 tissue-IHC caption).
How do I reduce diffuse or misleading DAB background?
First compare the stained section with a no-primary control and examine whether diffuse DAB persists outside intact cells (standard IHC practice). The selected workflow used 10% goat serum blocking, biotinylated goat anti-rabbit secondary, a streptavidin–biotin complex and DAB, so check each detection step for nonspecific deposition (PB9582 tissue-IHC caption). Include a peroxidase block as a general chromogenic IHC step, and assess endogenous biotin background when using a biotin-based detection system (standard IHC practice; PB9582 tissue-IHC caption). Titrate primary antibody and detection time on matched sections before interpreting widespread weak color as EIF6, whose tissue profile includes broad cytoplasmic and nuclear expression (standard IHC practice; HPA tissue IHC profile).
What is a defensible way to quantify EIF6 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then use an H-score or percent positive cells for intensity-based comparisons (standard IHC practice). Report nuclear and cytoplasmic results separately because both locations are supported for EIF6; include nucleolar signal in a stated nuclear scoring rule if assessed (UniProt P56537 subcellular location; HPA tissue IHC profile; standard IHC practice). Normalize cell-based scores to the number of evaluable cells in the same compartment and region, or report positive-cell density per mm² of viable tissue (standard IHC practice). Hold retrieval, DAB development, image acquisition and threshold rules constant, and exclude damaged edges and necrotic regions before comparing sections (standard IHC practice).
Which findings make an apparent EIF6-positive cell or region unconvincing?
Treat staining confined to tissue edges, folds or necrotic regions cautiously, especially when nearby intact cells lack the same pattern (standard IHC practice). Check a no-primary control for residual endogenous enzyme or detection-system color before assigning DAB deposits to EIF6 (standard IHC practice). A wholly membrane-restricted pattern needs further verification because EIF6 has no transmembrane segment and is annotated in cytoplasm and nucleus or nucleolus (UniProt P56537 topology and subcellular location). Compare cell identity and compartment with an appropriate positive region: HPA reports high signal in colon glandular cells and undetected signal in adipocytes, while its broader profile spans many tissues (HPA tissue IHC: colon glandular cells, adipocytes and profile).
Boster reagents
Best EIF6 / Eukaryotic translation initiation factor 6 IHC Antibodies
Anti-EIF6 antibodies have paraffin-section IHC images from human, mouse, and rat tissues (catalog IHC captions). PB9582 also lists ICC, but no IF image is supplied (PB9582 catalog applications and IF image alts).
Real IHC data
Anti-integrin beta 4 binding protein/EIF6 Antibody ®
Cat # PB9582
Real IHC data
Anti-EIF6 Antibody ® (monoclonal, 2I11)
Cat # M03581
Real IHC data
Anti-integrin beta 4 binding protein/EIF6 Antibody ®
Cat # PA2031
PB9582 shows mouse intestine, rat testis, and human intestinal cancer paraffin sections (PB9582 IHC captions); M03581 shows human intestinal, mammary, and lung cancers and rat spleen paraffin sections (M03581 IHC captions). PA2031 shows human intestinal cancer paraffin sections and human placenta frozen sections (PA2031 IHC captions).
Which to pick: For paraffin tissue IHC, choose PB9582 for citrate retrieval at pH 6 (PB9582 IHC caption), or monoclonal M03581 for EDTA retrieval at pH 8 (M03581 catalog clone and IHC caption); PA2031 also has a paraffin-section image (PA2031 IHC caption). For IF/ICC, PB9582 is the only SKU listing ICC (catalog applications), but it has no IF image (PB9582 IF image alts). All three list human, mouse, and rat reactivity for cross-species planning (catalog reactivity); their IHC captions do not report the fixative (catalog IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P56537 (IF6_HUMAN, Eukaryotic translation initiation factor 6).
- Human Protein Atlas. EIF6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. EIF6 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. EIF6 antibody validation summary (1 antibodies).
- Eukaryotic translation initiation factor 6 overexpression plays a major role in the translational control of gallbladder cancer. Journal of cancer research and clinical oncology 2019 — PMC6800842.
- Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC). Cancers 2021 — PMC8616251.
- Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Molecular cancer 2008 — PMC2383925.
- Process of Hypertrophic Scar Formation: Expression of Eukaryotic Initiation Factor 6. Chinese medical journal 2015 — PMC4736889.
- PubMed PMID:9405604 — UniProt-cited evidence.
- PubMed PMID:9374518 — UniProt-cited evidence.
- PubMed PMID:11290417 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EIF6 (Eukaryotic translation initiation factor 6). Expected localisation Cytoplasmic and nuclear in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9582). Validated IHC antibody: PB9582 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Mouse Intestine tissue; fixative not specified (datasheet PB9582); 2. Fixation: Image fixative and duration unreported (datasheet PB9582); 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: Citrate pH 6 , 20 min (datasheet PB9582); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9582); 8. Primary antibody: Rabbit anti-EIF6, 0.5-1μg/ml (datasheet PB9582); 9. Primary incubation: Overnight at 4 °C (datasheet PB9582). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB9582); verify before use.; Caveat: Adipocytes and myocytes have no detected staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unknown (UniProt); Regulation: Colon carcinoma exceeds normal colon (UniProt). Evidence: UniProt P56537, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet PB9582, PMC8616251, PMC4736889. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EIF6/immunohistochemistry-ihc-guide.