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EEF1A2 / Elongation factor 1-alpha 2 · IHC design guide
Design Immunohistochemistry for EEF1A2
Plan EEF1A2 paraffin-section IHC using neuronal cells with high staining as a positive control (HPA tissue IHC). Assess cytoplasmic staining in neurons and skeletal myocytes, while accounting for the antibody’s potential to target proteins from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EEF1A2 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 in neurons and skeletal myocytes (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic neuronal and skeletal myocyte staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A03304) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Adipose tissue+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 | Antibody may target proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Brain, heart, skeletal muscle expression (UniProt) | |
| Isoform / epitope | No isoforms listed; annotated chain spans residues 2–463 (UniProt) |
Section 1
Recommended EEF1A2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A03304) is followed by four published EEF1A2 IHC protocols (PMC11551808; PMC6586289; PMC1236916; PMC8007567).
| Sample | Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03304) |
| Fixation | Image fixative and duration unreported (datasheet A03304); 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 6.0 (datasheet A03304); 20 min, 95–100 °C (standard) |
| 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-EEF1A2, 1:50-1:200 (datasheet A03304) |
| 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 | EEF1A2-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in neuronal cells and skeletal myocytes. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6.0 (datasheet A03304); compare the published conditions when optimizing tissue staining (PMC11551808; PMC6586289; PMC1236916; PMC8007567).
Section 2
What Is the Expected EEF1A2 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic staining in neuronal cells and skeletal myocytes (HPA: tissue IHC profile). HPA reports cytosolic localisation by ICC-IF (HPA: subcellular localisation), while UniProt lists an endoplasmic reticulum membrane association without a transmembrane segment (UniProt Q05639: subcellular location and topology). Interpret the pattern cautiously: HPA rates tissue IHC Approved but reports low staining-to-RNA consistency and possible detection of protein from more than one gene (HPA: reliability description).
| Neuronal cytoplasm stains strongly in caudate, cerebral cortex or hippocampus (HPA: High in neuronal cells at these sites). | This matches the reported tissue pattern (HPA: tissue IHC). Judge the neuronal compartment and surrounding cells separately; a uniformly coloured section cannot establish cell-specific localisation (standard IHC practice). |
| Signal appears confined to nuclei or forms a sharp cell-surface outline, with little cytoplasmic staining. | That differs from the reported cytoplasmic tissue pattern and mainly cytosolic ICC-IF pattern (HPA: tissue IHC and subcellular localisation). Check morphology, counterstain and detection controls before interpreting it as EEF1A2 (standard IHC practice). |
| Adipocytes in adipose tissue stain strongly, or broad staining replaces a neuronal pattern (HPA: Not detected in adipocytes). | Treat the result as unexpected relative to the sampled HPA pattern (HPA: tissue IHC). Cross-reactivity or endogenous detection activity are possibilities to investigate, not diagnoses from appearance alone (standard IHC practice). |
| Colour spreads across stroma or empty regions and obscures cell boundaries. | Diffuse background prevents a reliable cell-specific call (standard IHC practice). Review blocking, washes and detection controls; HPA tissue levels do not identify the cause of background in a new assay (HPA: tissue IHC). |
| No signal appears in morphologically intact neuronal cells from a selected positive section. | This conflicts with High neuronal staining reported for several brain regions (HPA: tissue IHC). First assess the positive control and assay conditions; one negative run does not establish absence of EEF1A2 (standard IHC practice). |
💡Expected EEF1A2 appearanceCall a result positive when neuronal cytoplasm is clearly stained, often strongly in HPA-listed brain regions, or when skeletal myocyte cytoplasm is stained at a reported Medium level; isolated nuclear, cell-surface or diffuse background colour is suspect (HPA: tissue IHC; standard IHC practice).
| Which cells offer reference patterns? | Caudate, cerebral cortex and hippocampus show High neuronal staining; skeletal myocytes and cardiomyocytes show Medium staining (HPA: tissue IHC). Match the scored cell type to the reference, since a tissue name alone does not define every cell's expected result (standard IHC practice). |
| How should a negative reference be used? | HPA reports no detection in adipocytes from adipose tissue and glandular cells from appendix (HPA: tissue IHC). These are observations for specified cells; they do not prove that every cell in those organs is negative (HPA: tissue IHC). |
| What does localisation evidence support? | Tissue staining is described as cytoplasmic and ICC-IF localisation as mainly cytosolic (HPA: tissue IHC and subcellular localisation). UniProt also lists endoplasmic reticulum membrane localisation but no transmembrane segment (UniProt Q05639: location and topology); these records do not specify a distinctive membrane outline in paraffin IHC. |
| How firm is antibody-based interpretation? | Three listed antibodies have Approved IHC status: HPA051759, HPA056990 and CAB034019 (HPA: antibody validation). The tissue profile still carries low staining-to-RNA consistency and a multi-gene detection caution (HPA: reliability description); resolve unexpected patterns with controls or independent evidence (standard IHC practice). |
| Is target-specific fixation sensitivity known? | No target-specific fixation effect is supplied by the UniProt or HPA records in this guide (UniProt Q05639; HPA: tissue IHC). Retrieval conditions may be checked during general paraffin IHC optimization, without assuming that EEF1A2 has a documented fixation-sensitive epitope (standard IHC practice). |
| Can IF/ICC inform this IHC reading? | Yes: HPA reports an Approved, mainly cytosolic ICC-IF location and lists ICC approval for HPA051759, HPA053862, HPA056990 and CAB034019 (HPA: subcellular localisation and antibody validation). Use that localisation as supporting context; IF/ICC workflow belongs to its own guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive brain section has no neuronal colour (HPA: High in neuronal cells). | An IHC workflow or detection failure is possible; the slide alone cannot identify which step failed (standard IHC practice). | Check control tissue integrity, antibody application, retrieval settings and chromogen development against the assay record (standard IHC practice). |
| Neuronal nuclei dominate while cytoplasm is faint. | The distribution conflicts with cytoplasmic tissue staining and mainly cytosolic ICC-IF localisation (HPA: tissue IHC and subcellular localisation). | Recheck cell boundaries and counterstain; compare a positive control and detection-only control before scoring nuclei (standard IHC practice). |
| Adipocytes stain in a proposed negative reference (HPA: Not detected in adipocytes). | Cross-reactivity or endogenous detection activity may contribute; HPA warns that staining may target protein from more than one gene (HPA: reliability description). | Run appropriate detection controls and compare the IHC-validated antibody's pattern with independent evidence where available (standard IHC practice). |
| Brown signal covers stroma and masks cellular detail. | Non-specific reagent binding, insufficient washing or detection background may obscure localisation (standard IHC practice). | Inspect detection-only controls, blocking, wash steps and chromogen development; score only interpretable cellular staining (standard IHC practice). |
| Skeletal myocytes stain less intensely than neurons. | That difference can fit the reference: myocytes are Medium and neurons in several brain regions are High (HPA: tissue IHC). | Score each cell type against its own reported pattern and the run's controls before calling the muscle result a failure (HPA: tissue IHC; standard IHC practice). |
| A convincing cytoplasmic pattern appears in an unexpected cell type. | HPA's cell-specific observations and multi-gene detection caution limit target attribution from colour alone (HPA: tissue IHC and reliability description). | Record the cell type and compartment, examine controls, and seek independent target evidence before assigning the signal to EEF1A2 (standard IHC practice). |
Experiment evidence
IHC & IF images showing EEF1A2 staining
A03304 · IHCImmunohistochemistry of paraffin-embedded rat brain using EEF1A2 Rabbit pAb at dilution of 1:100 .Perform high pressure antigen retrieval wi
A03304 · IHCImmunohistochemistry of paraffin-embedded human brain using EEF1A2 Rabbit pAb at dilution of 1:100 .Perform high pressure antigen retrieval
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101210-EEF1A2/tissue/cerebral+cortex#imid_8852596
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101210-EEF1A2/tissue/colon#imid_8852522
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101210-EEF1A2/tissue/liver#imid_8852515
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101210-EEF1A2/tissue/kidney#imid_8852516
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody CAB034019; green = EEF1A2, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody CAB034019; green = EEF1A2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EEF1A2 IHC & IF
🧪Run caudate first and look for staining in neuronal cells (HPA: High in caudate neuronal cells). Use bronchus respiratory epithelial cells as a negative comparator (HPA: Not detected); on the caudate slide, assess neighboring non-neuronal cells for background staining without assuming they lack EEF1A2.
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EEF1A2 in MCF-7, U2OS, PC-3, Hep-G2, hTERT-RPE1, ASC52telo, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls for the rabbit polyclonal catalog antibody, plus EEF1A2 knockout material as a biological negative (A03304 caption: rabbit pAb; standard IHC practice). For chromogenic brain IHC, block endogenous peroxidase and inspect tissue pigment; for brain IF, check autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03304 paraffin-section caption does not state a fixative. Its rat-brain IHC image used high-pressure retrieval in 10 mM citrate at pH 6.0 and antibody at 1:100 (A03304 caption); use these as starting conditions, since retrieval dependency is unreported. No supplied comparison establishes that frozen sections or IF are easier; for IF/ICC, expect mainly cytosolic signal (HPA: approved cytosol localization) and assess brain lipofuscin autofluorescence (standard IF practice).
HPA tissue IHC evidence for EEF1A2
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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EEF1A2 IHC Tips
Troubleshoot EEF1A2 staining in paraffin sections by checking retrieval, cell type, cytoplasmic signal and antibody specificity before comparing scores.
What should I change when EEF1A2 staining is weak after retrieval?
Start with heat-mediated retrieval in 10 mM citrate buffer at pH 6.0 before chromogenic IHC (datasheet A03304). The selected paraffin-section rat brain image used high-pressure retrieval and a 1:100 primary dilution, giving a documented starting point for that antibody and tissue (caption A03304). If staining is weak, compare a modestly longer heating cycle with the current cycle on adjacent sections, keeping antibody dilution and detection constant (standard IHC practice). Include a known positive neuronal area and inspect tissue integrity, because excessive heating can damage sections and make any apparent gain difficult to interpret (HPA: high in cerebral cortex neuronal cells; standard IHC practice).
How can I troubleshoot weak staining when fixation history varies?
Target-specific sensitivity of EEF1A2 to fixative type or duration is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A03304). Record fixative, time in fixative, processing history and section age for each specimen before comparing staining intensity (standard IHC practice). Run sections with different histories in the same staining batch, using the same citrate pH 6.0 retrieval and detection conditions (datasheet A03304; standard IHC practice). If signal differs, test retrieval and primary dilution separately on adjacent sections, and report the fixation difference as a possible confounder rather than an established EEF1A2 effect (standard IHC practice).
Should I accept membrane-like or nuclear EEF1A2 staining in neuronal sections?
Evaluate neuronal cytoplasm first: tissue staining is described in neuronal cells, and the approved subcellular location is cytosol (HPA: cytoplasmic neuronal staining; HPA: cytosol approved). UniProt also lists an endoplasmic-reticulum membrane association but no transmembrane segment, so a membrane outline alone is insufficient evidence of specific staining (UniProt Q05639: location and topology). Compare the suspect pattern with adjacent neuronal cytoplasm at the same magnification, including a no-primary control to assess detection background (HPA: neuronal staining; standard IHC practice). If staining is exclusively nuclear or sharply outlines cells without cytoplasmic signal, reassess antibody specificity, retrieval and section artefacts before scoring it as EEF1A2 (standard IHC practice).
Can staining distinguish EEF1A2 from a related translation factor or epitope variant?
The supplied UniProt record lists 0 isoforms, but EEF1A2 carries multiple modified residues, including alternative methylation states at residue 36 (UniProt Q05639: isoforms and modified residues). Those annotations do not establish whether this antibody recognizes every modification state or distinguishes EEF1A2 from a related protein (UniProt Q05639; standard IHC interpretation). Check the antibody's stated immunogen and specificity evidence, then compare staining across documented positive and low-signal tissue compartments under identical conditions (standard IHC practice; HPA: high neuronal staining, low oral mucosa staining). Treat discordance cautiously because HPA reports low consistency with RNA expression and warns that its antibody targets protein from more than one gene (HPA: reliability description).
How should I investigate an EEF1A2 pattern seen by IF alongside chromogenic IHC?
Use IF as a complementary localization check, pairing EEF1A2 with a validated marker of the expected neuronal cell type when examining brain sections (HPA: neuronal cytoplasmic staining; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect unstained and single-label controls before interpreting overlap (standard IF practice). EEF1A2 has no transmembrane segment and is mainly cytosolic by approved imaging, so permeabilisation should provide access to the cytosolic epitope; its exact antibody epitope is unspecified (UniProt Q05639: topology; HPA: cytosol approved). Keep IF fixation and permeabilisation optimization separate from the catalog antibody's paraffin-section IHC result, whose fixative is unreported (caption A03304; standard IF practice).
What explains diffuse brown signal outside expected EEF1A2-positive cells?
First compare the brown deposit with expected cytoplasmic staining in neuronal cells or skeletal myocytes, rather than treating uniform section color as EEF1A2 (HPA: tissue profile). Include a no-primary control, block endogenous peroxidase, and check whether DAB develops in both test and control sections (standard chromogenic IHC practice). If background follows tissue folds, section edges or dried areas, repeat staining with even reagent coverage and thorough washes before changing primary concentration (standard IHC practice). If background persists only with primary antibody, titrate around the documented 1:100 rat brain starting dilution while keeping retrieval and development time matched (caption A03304; standard IHC practice).
How should I score EEF1A2 when sections contain mixed cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the eligible cell population before scoring: HPA describes high staining in several neuronal populations and medium staining in skeletal myocytes and cardiomyocytes (HPA: tissue IHC). For chromogenic sections, report the percentage of positive eligible cells and an intensity-weighted H-score, or positive-cell density per mm² when cell counts are appropriate (standard IHC practice). Normalize each measure to the number or area of the defined eligible cells, rather than total tissue area when unrelated compartments vary between sections (standard IHC practice). Apply one threshold, counterstain assessment and region-selection rule across batches, and report HPA's antibody cross-gene caution when interpreting absolute positivity (standard IHC practice; HPA: reliability description).
When is a strong brown signal unlikely to represent true EEF1A2?
A convincing result follows expected cytoplasmic staining in neuronal cells or skeletal myocytes, with the specific cell type and compartment recorded (HPA: tissue profile; HPA: cytosol approved). Isolated nuclear staining or a crisp cell-surface rim needs further validation because the approved location is cytosol and UniProt lists no transmembrane segment (HPA: subcellular; UniProt Q05639: topology). Exclude staining confined to section edges, folds or necrotic areas, and use a no-primary control to assess endogenous enzyme or detection-system color (standard chromogenic IHC practice). Even plausible cytoplasmic staining requires cautious attribution to EEF1A2 because HPA flags low RNA–protein consistency and antibody recognition of protein from more than one gene (HPA: reliability description).
Boster reagents
Best EEF1A2 / Elongation factor 1-alpha 2 IHC Antibodies
A03304 has IHC images of paraffin-embedded human, mouse, and rat brain (A03304 image captions). No IF data are supplied (catalog applications; IF image alts).
Real IHC data
Anti-Elongation factor 1-alpha 2 EEF1A2 Antibody
Cat # A03304
A03304 is listed for IHC and reactivity with human, mouse, and rat (catalog applications; reactivity). Its IHC captions show paraffin-embedded brain from all three species stained at 1:100 after high-pressure retrieval in 10 mM citrate buffer, pH 6.0 (A03304 image captions).
Which to pick: Choose A03304 for paraffin-section tissue IHC across human, mouse, and rat samples; its captions identify a rabbit polyclonal antibody and show IHC staining in each species (A03304 image captions). The catalog IHC range is 1:50–1:200, and the pictured sections used 1:100 with citrate retrieval; the fixative is unreported (catalog IHC dilution; A03304 image captions). For IF/ICC, this payload provides no listed IF application or IF figure for A03304 (catalog applications; IF image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q05639 (EF1A2_HUMAN, Elongation factor 1-alpha 2).
- Human Protein Atlas. EEF1A2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. EEF1A2 subcellular location (ICC-IF): Mainly localized to the cytosol..
- Human Protein Atlas. EEF1A2 antibody validation summary (4 antibodies).
- High expression of eukaryotic elongation factor 1-alpha-2 in lung adenocarcinoma is associated with poor prognosis. Pathology international 2024 — PMC11551808.
- Elevated levels of eEF1A2 protein expression in triple negative breast cancer relate with poor prognosis. PloS one 2019 — PMC6586289.
- Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours. BMC cancer 2005 — PMC1236916.
- EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling. British journal of cancer 2021 — PMC8007567.
- PubMed PMID:8354261 — UniProt-cited evidence.
- PubMed PMID:10950927 — UniProt-cited evidence.
- PubMed PMID:19054851 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EEF1A2 (Elongation factor 1-alpha 2). Expected localisation Cytoplasmic in neurons and skeletal myocytes (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A03304). Validated IHC antibody: A03304 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03304); 2. Fixation: Image fixative and duration unreported (datasheet A03304); 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 6.0 (datasheet A03304); 20 min, 95–100 °C (standard); 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-EEF1A2, 1:50-1:200 (datasheet A03304); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody may target proteins from more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms listed; annotated chain spans residues 2–463 (UniProt); Regulation: Brain, heart, skeletal muscle expression (UniProt). Evidence: UniProt Q05639, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol., Datasheet A03304, PMC11551808, PMC6586289. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EEF1A2/immunohistochemistry-ihc-guide.