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EIF6 / Eukaryotic translation initiation factor 6 · IHC design guide
Design Immunohistochemistry for EIF6
Plan EIF6 staining in paraffin sections around cytoplasmic and nuclear signal (HPA tissue IHC). Use high-staining glandular cells and unstained adipocytes or myocytes as reference populations (HPA tissue IHC), and score the two compartments separately (standard IHC practice).
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 tissue staining (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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9582) | |
| Caveat | Broad expression limits whole-tissue negative controls (HPA tissue IHC) | |
| Regulation | Higher in colon carcinoma than normal colon (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage needs checking (UniProt) |
Section 1
Recommended EIF6 IHC & IF Protocols
Compare the catalog antibody's citrate pH 6 IHC protocol (datasheet PB9582) with four published EIF6 IHC methods (PMC6800842; PMC8616251; PMC2383925; 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 heat-mediated citrate pH 6 retrieval (datasheet PB9582). The OSC study also used boiled citrate pH 6 (PMC2383925); the scar study used Proteinase K (PMC4736889).
Section 2
What Is the Expected EIF6 Staining Pattern?
EIF6 should appear in the cytoplasm and nucleus of many tissue cells (HPA: tissue IHC profile), with nucleolar localisation also reported (UniProt P56537: subcellular location). It has no transmembrane segment (UniProt P56537: topology). The HPA tissue pattern has Enhanced reliability, reflecting consistency between staining and RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic and nuclear staining in glandular epithelium, with strong signal in colon, appendix or duodenum (HPA: High in glandular cells). | This fits the reported tissue pattern (HPA: cytoplasmic and nuclear expression in most tissues). Score the glandular cells and compartments separately; a single field need not represent the whole section (general IHC practice). |
| Predominantly membranous or extracellular staining, without convincing cytoplasmic or nuclear signal. | That distribution is unexpected for EIF6 (UniProt P56537: cytoplasm and nucleus; no transmembrane segment). Check morphology and staining controls before calling it specific (general IHC practice). |
| Strong staining confined to adipocytes or muscle cells while nearby expected cells are unstained. | Those cells are reported as Not detected (HPA: adipocytes, skeletal myocytes and smooth muscle cells). Consider cross-reactivity or endogenous detection activity, then compare an appropriate negative control (general IHC practice). |
| A uniform haze obscures nuclei, cytoplasm and tissue boundaries. | Diffuse background cannot establish EIF6 localisation. Assess blocking, antibody concentration, washes and detection controls before interpreting cellular staining (general IHC practice). |
| No signal in colon glandular cells despite intact tissue morphology. | Colon glandular cells are reported High (HPA: colon tissue IHC). Check the staining run and a known-positive control first; a negative field alone does not establish EIF6 absence (general IHC practice). |
💡Expected EIF6 appearanceA convincing positive shows cytoplasmic and nuclear staining in appropriate cells, potentially strong in colon glandular cells (HPA: tissue IHC profile; High in colon glandular cells), while isolated membrane-like signal or staining limited to HPA-negative adipocytes is suspect (UniProt P56537: topology; HPA: adipocytes Not detected).
| Tissue and cell selection | Colon, appendix and duodenum glandular cells are reported High, making them useful positive comparators (HPA: tissue IHC). Adipocytes and skeletal or smooth muscle cells are reported Not detected (HPA: tissue IHC). |
| Compartment and topology | EIF6 shuttles between cytoplasm and nucleus/nucleolus and lacks a transmembrane segment (UniProt P56537). Do not require equal staining in both compartments in every cell; evaluate the observed pattern against controls (general IHC practice). |
| Evidence and antibody validation | The tissue profile is Enhanced, and HPA040873 has Enhanced IHC validation (HPA: tissue reliability; antibody validation). These support the reported pattern but do not establish specificity for every experimental sample (general IHC practice). |
| Normal tissue context | Kidney tubular cells are High in HPA tissue IHC, whereas UniProt describes kidney among tissues with the lowest protein levels in its tissue-specificity account (HPA: kidney; UniProt P56537: tissue specificity). Preserve the assay-specific context when comparing them. |
| Isoforms and modifications | UniProt lists two isoforms and several phosphorylation sites (UniProt P56537: isoforms; modified residues). The supplied sources do not map the antibody epitope, so they do not establish isoform or phosphorylation-dependent IHC staining. |
| IF/ICC: what localisation is expected? | HPA reports supported nucleoplasmic localisation in ICC-IF (HPA: subcellular summary). UniProt also reports cytoplasmic and nucleolar localisation (UniProt P56537: subcellular location); the ICC-IF result need not duplicate every tissue IHC compartment. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells show no staining. | A failed staining or detection step is possible; colon, appendix and duodenum glandular cells are reported High (HPA: tissue IHC). | Confirm the positive control, antibody identity, retrieval and detection steps using the validated IHC workflow (general IHC practice). Do not infer target-specific fixation sensitivity from this result. |
| Only the tissue edge or damaged regions stain intensely. | The distribution may reflect tissue handling or nonspecific staining rather than cellular localisation (general IHC practice). | Judge intact central regions and repeat with appropriate staining controls before scoring EIF6 (general IHC practice). |
| Adipocytes or myocytes appear strongly positive. | This conflicts with their Not detected HPA tissue pattern; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Review cell identity, a negative control and detection-background controls; compare with expected glandular-cell staining (general IHC practice; HPA: glandular cells High). |
| A membrane outline dominates the signal. | EIF6 has no transmembrane segment and is reported in cytoplasm and nucleus (UniProt P56537: topology; subcellular location). | Check whether cytoplasmic or nuclear signal is present and compare the suspect outline with negative controls (general IHC practice). |
| Diffuse chromogen obscures compartment boundaries. | Excess background can prevent interpretation of localisation (general IHC practice). | Review blocking, antibody concentration, washing and detection controls, then score only resolvable cells (general IHC practice). |
| Nuclear signal varies between fields while cytoplasmic signal remains. | EIF6 shuttles between cytoplasm and nucleus/nucleolus, so unequal compartment intensity alone does not prove an artefact (UniProt P56537: subcellular location). | Compare intact cells, tissue regions and run controls before assigning a localisation change (general IHC practice). |
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: glandular cells should stain (HPA: High in colon glandular cells). Use skeletal muscle as the negative tissue (HPA: Not detected in myocytes); smooth muscle cells within the colon section, if present, should remain at background (HPA: Not detected in smooth muscle cells).
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 and a concentration-matched rabbit IgG isotype control (PB9582 caption: rabbit primary antibody), plus EIF6 knockout material or an immunizing-peptide block if available. Quench endogenous peroxidase and block endogenous biotin when using the caption’s biotin-based DAB detection (PB9582 caption: biotinylated secondary, streptavidin–biotin complex and DAB).
⚠️Feasibility: No supplied source reports an EIF6-specific fixation window or fixation effect; the selected PB9582 tissue-IHC caption does not state the fixative (PB9582 caption: fixative not stated). The paraffin-section example used heat retrieval in citrate buffer at pH 6 for 20 minutes (PB9582 caption); the supplied evidence does not establish whether frozen sections or IF are easier. In colon, interpret glandular-cell staining separately from luminal material and smooth-muscle background (HPA: High in colon glandular cells; Not detected in smooth muscle cells).
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
Use the documented paraffin section workflow as a starting point, then assess EIF6 staining by cell type and compartment.
Which retrieval conditions should I start with for EIF6 staining in paraffin sections?
Use heat-mediated citrate pH 6 antigen retrieval for paraffin EIF6 IHC (datasheet PB9582). The selected tissue image used 20 minutes of retrieval before staining with 1 μg/mL primary antibody overnight at 4°C (PB9582 tissue-IHC caption). If staining is weak, first check heating consistency and section adhesion; then compare a small set of sections with an alternative retrieval buffer, such as EDTA pH 9, while keeping detection conditions fixed (standard IHC practice). Judge any improvement by cellular staining and preservation of tissue structure, since EIF6 can appear in both cytoplasm and nucleus (HPA tissue IHC; UniProt P56537 subcellular location).
How should I troubleshoot weak EIF6 staining when fixation details are incomplete?
The selected paraffin tissue-IHC caption does not state a fixative, so EIF6 sensitivity to fixation is unknown for this antibody (PB9582 tissue-IHC caption). Record the fixative, fixation duration, tissue thickness and processing history for each specimen before comparing staining intensity (standard IHC practice). On matched sections, keep citrate pH 6 retrieval for 20 minutes and the 1 μg/mL primary incubation constant while checking whether weak staining follows a processing batch (PB9582 tissue-IHC caption; standard IHC practice). Compare intact cells and tissue preservation alongside staining; neither HPA expression patterns nor EIF6 molecular features establish a target-specific fixation effect (HPA tissue IHC; UniProt P56537 record).
Is nuclear EIF6 staining expected, or should I score only cytoplasmic signal?
Score nuclear and cytoplasmic EIF6 separately: tissue IHC reports both compartments in most tissues, and EIF6 shuttles through the nucleus and nucleolus (HPA tissue IHC; UniProt P56537 subcellular location). Nucleoplasmic localisation is also supported by cell imaging, although that observation does not set an IHC staining threshold (HPA subcellular). With a hematoxylin counterstain, assess nuclear boundaries and adjacent cytoplasm in intact cells rather than assigning diffuse brown deposits to either compartment (standard IHC practice). Record the fraction and intensity in each compartment using the same scoring rules across sections, and compare against expected glandular or tubular cells in the available tissue reference (HPA tissue IHC; standard IHC practice).
Can this stain distinguish EIF6 isoforms or phosphorylation states?
Do not assign a chromogenic signal to a particular EIF6 isoform: 2 isoforms are listed, but the supplied antibody evidence gives no epitope map or isoform selectivity (UniProt P56537 isoforms; PB9582 tissue-IHC caption). EIF6 also has annotated phosphorylation sites, including residues 113, 165 and 166, without evidence that this stain reports any one modification (UniProt P56537 modified residues; PB9582 tissue-IHC caption). When experiments require those distinctions, obtain epitope and modification-specific validation before interpreting staining differences (standard IHC practice). Keep retrieval, primary concentration and detection constant across comparison sections so technical variation does not masquerade as an isoform or phosphorylation effect (standard IHC practice).
How can I investigate EIF6 localisation by IF after seeing ambiguous IHC staining?
Treat IF as a separate assay and establish its antibody and fixation conditions independently of the paraffin IHC image (PB9582 tissue-IHC caption; standard IF practice). Multiplex EIF6 with a validated marker for the expected cell population, such as glandular cells in colon or tubular cells in kidney, and inspect nuclear and cytoplasmic channels separately (HPA tissue IHC; UniProt P56537 subcellular location). Choose fluorophores after checking tissue autofluorescence and include single-channel controls to assess bleed-through (standard IF practice). EIF6 has no transmembrane segment, so choose permeabilisation that permits access to intracellular epitopes while preserving compartment boundaries; its exact antibody epitope and IF fixation sensitivity are unspecified here (UniProt P56537 topology; PB9582 tissue-IHC caption).
What should I adjust when EIF6 DAB staining appears widespread and nonspecific?
First compare stained cells with a no-primary control and inspect whether brown signal follows tissue edges, folds or damaged areas (standard IHC practice). The documented workflow used 10% goat serum, 1 μg/mL rabbit primary overnight at 4°C, a biotinylated secondary and DAB detection (PB9582 tissue-IHC caption). If background persists, adjust blocking and primary concentration on matched sections, and check endogenous peroxidase and biotin contributions before changing the interpretation of widespread staining (standard IHC practice). Widespread cellular signal is plausible because tissue IHC reports cytoplasmic and nuclear EIF6 in most tissues, but diffuse deposits without cell boundaries need independent control evidence (HPA tissue IHC; standard IHC practice).
How should I quantify EIF6 when both nuclear and cytoplasmic staining vary? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear and cytoplasmic staining in separate fields before calculating a specimen summary (HPA tissue IHC; UniProt P56537 subcellular location; standard IHC practice). For each compartment, report the percentage of positive cells and an H-score from 0–300, calculated by summing the percentage at each 0–3 intensity grade multiplied by that grade (standard IHC practice). Normalise counts to eligible intact cells, or positive-cell density to analysed tissue area in mm², and exclude folds and necrosis consistently (standard IHC practice). Keep imaging, threshold and scoring rules fixed across specimens; use glandular or tubular reference populations only when they are present in the sections being assessed (HPA tissue IHC; standard IHC practice).
How can I distinguish convincing EIF6 staining from a tissue or detection artefact?
A convincing result has cell-bounded cytoplasmic and/or nuclear staining, consistent with tissue IHC and EIF6 shuttling between those compartments (HPA tissue IHC; UniProt P56537 subcellular location). Compare the pattern with the sampled cell types: glandular cells in colon and tubular cells in kidney are reported high, whereas adipocytes and skeletal myocytes are reported undetected (HPA tissue IHC). Treat staining confined to section edges, necrotic regions or damaged cells as suspect, and check a no-primary control for detection-related deposits (standard IHC practice). Investigate granular signal that survives primary omission through peroxidase and biotin controls before calling it EIF6, because the documented chromogenic workflow uses biotin-based detection and DAB (PB9582 tissue-IHC caption; standard IHC practice).
Boster reagents
Best EIF6 / Eukaryotic translation initiation factor 6 IHC Antibodies
Catalog anti-EIF6 antibodies have IHC images from human, mouse and rat tissues (catalog IHC captions). PB9582 also lists ICC, but no IF image is supplied (PB9582 applications; catalog IF images).
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 has paraffin-section IHC images from mouse intestine, rat testis and human intestinal cancer, and lists ICC (PB9582 image captions; PB9582 applications). M03581 has paraffin-section IHC images from human cancers and rat spleen; PA2031 has a human intestinal cancer paraffin-section IHC image (M03581 image captions; PA2031 image caption).
Which to pick: For tissue IHC, choose PB9582 when citrate retrieval at pH 6 matches the planned workflow, or monoclonal M03581 when EDTA retrieval at pH 8 is preferred (PB9582 image caption; M03581 image caption; M03581 clone 2I11). For ICC, PB9582 is the listed option; its catalog supplies no IF image, so IF performance is unshown (PB9582 applications; catalog IF images). For cross-species tissue IHC, PB9582 has images from human, mouse and rat sections; its caption identifies paraffin sections but does not report the fixative (PB9582 image 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 tissue staining (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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9582); Caveat: Broad expression limits whole-tissue negative controls (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage needs checking (UniProt); Regulation: Higher in colon carcinoma than normal colon (UniProt). Evidence: UniProt P56537, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet PB9582, PMC6800842, PMC8616251. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EIF6/immunohistochemistry-ihc-guide.