EIF4A1 / Eukaryotic initiation factor 4A-I · IHC design guide

Design Immunohistochemistry for EIF4A1

Plan EIF4A1 paraffin IHC around the cytoplasmic staining reported in several tissues (HPA tissue IHC). This guide uses catalog antibody conditions for paraffin sections (datasheet A03922-2) and flags the uncertain reliability of the tissue staining profile (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for EIF4A1 (IHC for EIF4A1): expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC), antibody A03922-2, validated IHC image, and IHC protocol steps — Printable **EIF4A1** IHC protocol sheet — expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC), antibody A03922-2, controls and protocol steps. Open the full EIF4A1 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

EIF4A1 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 several tissues (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining across several tissue cell types (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A03922-2)
	Positive control ⓘ	Bone marrow+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03922-2)
	Caveat	Staining–RNA concordance is low; reliability is uncertain (HPA tissue IHC)
	Regulation	Low tissue specificity (HPA tissue RNA)
	Isoform / epitope	Three isoforms; epitope effects are undetermined (UniProt)

Section 1

Recommended EIF4A1 IHC & IF Protocols

The catalog antibody protocol (datasheet: A03922-2) is followed by three published EIF4A1 IHC protocols (PMC9441068; PMC6857661; PMC10195147).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human colon cancer tissues; fixative not specified (datasheet A03922-2)
Fixation	Image fixative and duration unreported (datasheet A03922-2); 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 A03922-2)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03922-2)
Primary antibody	Rabbit anti-EIF4A1, 0.5-1μg/ml (datasheet A03922-2)
Primary incubation	Overnight at 4 °C (datasheet A03922-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03922-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	EIF4A1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control.

💡Decision noteStart with citrate pH 6 heat retrieval (datasheet: A03922-2); the TMA study used a different retrieval buffer at 95 °C for 30 min (PMC9441068).

Section 2

What Is the Expected EIF4A1 Staining Pattern?

EIF4A1 is primarily cytoplasmic, with perinuclear, cell membrane and stress granule localisation also annotated (UniProt P60842). In paraffin sections, expect cytoplasmic staining in the cell populations reported by HPA, including colon glandular cells and bone marrow hematopoietic cells (HPA tissue IHC). HPA rates its tissue IHC pattern Uncertain because antibody staining and RNA expression have low consistency; interpret each result with controls (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic signal in colon glandular cells, placental trophoblastic cells or bone marrow hematopoietic cells, with limited background (HPA tissue IHC).	This matches cell populations scored High by HPA and the expected compartment (HPA tissue IHC; UniProt P60842). Record the fraction and intensity of stained cells separately: a positive population need not make every cell equally dark. HPA's Uncertain reliability means agreement is supportive, not confirmation of antibody specificity (HPA tissue IHC).
Predominantly nuclear staining, with little cytoplasmic signal, in an otherwise well-preserved section.	Nuclear enrichment does not match the reported cytoplasmic tissue pattern or approved cytosolic ICC-IF localisation (HPA tissue IHC; HPA subcellular ICC-IF). Consider nonspecific staining, detection background or an interpretation error; compare the positive control and the no-primary control before assigning a nuclear EIF4A1 pattern (general IHC practice).
Strong signal in adipocytes or adrenal gland glandular cells while expected positive cells stain weakly (HPA tissue IHC).	HPA reports those specific cell populations as Not detected, so this reversal warrants checking cell identification, cross-reactivity and endogenous detection activity (HPA tissue IHC; general IHC practice). It does not establish that the whole tissue is EIF4A1-negative, and the HPA tissue pattern remains Uncertain (HPA tissue IHC).
Diffuse color across nuclei, stroma and extracellular spaces, obscuring cell boundaries.	A widespread haze cannot be scored as the cell-associated cytoplasmic pattern reported for EIF4A1 (HPA tissue IHC). Compare a no-primary control, review blocking and washing, and check whether the detection system produces signal without primary antibody (general IHC practice). Score only interpretable cells after background is controlled.
No stain in colon glandular cells or bone marrow hematopoietic cells, despite preserved morphology (HPA tissue IHC).	These are HPA High populations, so a blank result calls for a control-based check of antibody performance, detection and the general IHC workflow (HPA tissue IHC; general IHC practice). A single negative section does not prove EIF4A1 absence; HPA's tissue IHC assessment is itself Uncertain (HPA tissue IHC).

💡Expected EIF4A1 appearanceCall a result positive when cell-associated cytoplasmic staining is clear in an HPA High population such as colon glandular cells, with low surrounding background; isolated nuclear signal or diffuse color without cell boundaries is suspect (HPA tissue IHC; UniProt P60842; general IHC practice).

How each factor affects the staining
Compartment and topology	UniProt places EIF4A1 in cytoplasm and the perinuclear region, and also annotates cell membrane and stress granules; it lists no transmembrane segment (UniProt P60842). Expect the main routine tissue readout to be cytoplasmic (HPA tissue IHC). Membrane-associated signal should be judged with the whole cellular pattern, not treated as proof of an integral membrane protein (UniProt P60842 topology).
Choice of tissue and cell population	HPA scores colon, rectum and stomach glandular cells, placental trophoblastic cells, bone marrow hematopoietic cells and tonsillar squamous epithelial cells High; bronchial respiratory epithelial cells and appendix lymphoid tissue are Medium (HPA tissue IHC). Select a documented positive population for a control and score that population rather than averaging the entire section (general IHC practice).
Strength of the tissue evidence	The HPA tissue profile says cytoplasmic expression in several tissues, but assigns Uncertain reliability because staining and RNA expression show low consistency and external verification is pending (HPA tissue IHC). Its antibody listing gives CAB011689 an Uncertain IHC status; HPA068286 has ICC approval and no listed IHC status (HPA antibodies). Treat intensity rankings as comparison points, not validated cutoffs.
Isoforms and antibody epitope	UniProt lists three isoforms and helicase regions at residues 63–234 and 245–406 (UniProt P60842). The supplied evidence gives no epitope for the antibody being scored, so it cannot establish which isoforms that antibody detects or whether an unusual tissue result reflects isoform selection. Check the antibody's documented target region if interpreting discordant results (general IHC practice).
IF/ICC Q&A: What pattern should fluorescence show?	Predominantly cytosolic signal: HPA approves the main cytosol localisation in ICC-IF images from CACO-2, U-251MG and U2OS (HPA subcellular ICC-IF). HPA cautions that this assignment is based on antibodies targeting proteins from multiple genes, so it is supporting localisation evidence rather than independent validation of this IHC result (HPA subcellular ICC-IF).
Antigen retrieval and detection conditions	Retrieval, blocking and detection controls are general paraffin IHC workflow considerations (general IHC practice). The supplied UniProt and HPA records give no EIF4A1-specific retrieval setting or fixation sensitivity; neither tissue intensity nor protein topology establishes one (UniProt P60842; HPA tissue IHC). Evaluate any workflow change against a documented positive population and a no-primary control (general IHC practice).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells are blank.	A workflow or detection failure is possible; HPA labels those cells High but the tissue IHC evidence is Uncertain (HPA tissue IHC).	Confirm the intended cell population, then review the IHC-validated antibody's documented conditions, retrieval, detection reagents and positive-control performance (general IHC practice). Avoid calling the specimen negative until the control stains.
Only nuclei appear strongly positive.	This conflicts with the cytoplasmic tissue profile and approved cytosol ICC-IF localisation (HPA tissue IHC; HPA subcellular ICC-IF).	Compare with the no-primary control, inspect cytoplasmic staining in a documented positive population, and review counterstain and detection background (general IHC practice). Report unresolved nuclear signal as atypical.
Adipocytes stain strongly.	HPA reports adipocytes as Not detected; misidentified cells, cross-reactivity or endogenous detection activity may explain the signal (HPA tissue IHC; general IHC practice).	Verify the stained cell type and compare a no-primary control and a documented HPA High population in the same staining run (HPA tissue IHC; general IHC practice).
Brown precipitate or haze covers tissue regardless of cell type.	Diffuse staining is inconsistent with HPA's cell-associated cytoplasmic profile and can arise from detection background (HPA tissue IHC; general IHC practice).	Check the no-primary control; review blocking, washes and chromogen development using the detection system's instructions (general IHC practice). Reassess localisation only where cell boundaries remain clear.
A medium or low HPA population looks weaker than a high population.	HPA reports different levels across cell populations: bronchial respiratory epithelial cells are Medium, while colon glandular cells are High (HPA tissue IHC).	Score each named cell population separately and compare intensity within the same staining run (general IHC practice). Keep HPA's Uncertain tissue reliability in the interpretation (HPA tissue IHC).
IHC and ICC-IF localisation appear discordant.	HPA ICC-IF supports cytosol localisation but cautions that its assignment draws on antibodies targeting proteins from multiple genes; tissue IHC reliability is Uncertain (HPA subcellular ICC-IF; HPA tissue IHC).	Document the antibody used in each application, compare the compartment seen in each image, and assess each application's controls before inferring a biological difference (general IHC/IF practice).

Source: UniProt P60842 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing EIF4A1 staining

IHC analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in paraffin-embedded section of human colon cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-EIF4A Antibody (A03922-2) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A03922-2 · IHCIHC analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in paraffin-embedded section of human colon cancer tissues. H

IHC analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in paraffin-embedded section of mouse intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-EIF4A Antibody (A03922-2) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A03922-2 · IHCIHC analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in paraffin-embedded section of mouse intestine tissues. Heat

IF analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-EIF4A Antibody (A03922-2) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03922-2 · IFIF analysis of EIF4A using anti-EIF4A antibody (A03922-2). EIF4A was detected in immunocytochemical section of U20S cell. Enzyme antigen ret

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-EIF4A1 Antibody (M03922-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M03922-2 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in a paraffin-embedded section of human placenta tissue. H

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in a paraffin-embedded section of human colonic adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-EIF4A1 Antibody (M03922-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M03922-2 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in a paraffin-embedded section of human colonic adenocarci

IF analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in an immunocytochemical section of HEP3B cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL mouse anti-EIF4A1 Antibody (M03922-2) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

M03922-2 · IFIF analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-2). EIF4A1 was detected in an immunocytochemical section of HEP3B cells. Enzyme ant

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-EIF4A1 Antibody (A03922-3) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A03922-3 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in paraffin-embedded section of human tonsil tissue. Heat

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in paraffin-embedded section of human Lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-EIF4A1 Antibody (A03922-3) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A03922-3 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in paraffin-embedded section of human Lung cancer tissue.

IF analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-EIF4A1 Antibody (A03922-3) overnight at 4°C. DyLight®488 conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03922-3 · IFIF analysis of EIF4A1 using anti-EIF4A1 antibody (A03922-3). EIF4A1 was detected in immunocytochemical section of A431 cells. Enzyme antigen

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in paraffin-embedded section of human gastric cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-EIF4A1 Antibody (M03922-1) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M03922-1 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in paraffin-embedded section of human gastric cancer tissu

IHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml mouse anti-EIF4A1 Antibody (M03922-1) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

M03922-1 · IHCIHC analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in paraffin-embedded section of human breast cancer tissue

IF analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in immunocytochemical section of CACO-2 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL mouse anti-EIF4A1 Antibody (M03922-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

M03922-1 · IFIF analysis of EIF4A1 using anti-EIF4A1 antibody (M03922-1). EIF4A1 was detected in immunocytochemical section of CACO-2 cells. Enzyme antig

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000161960-EIF4A1/tissue/cerebral+cortex#imid_3643584

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000161960-EIF4A1/tissue/colon#imid_3643510

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000161960-EIF4A1/tissue/liver#imid_3643527

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000161960-EIF4A1/tissue/kidney#imid_3643528

HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA068286; green = EIF4A1, blue = nucleus, red = microtubules.

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Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for EIF4A1 IHC & IF

🧪Run colon first and expect staining in its glandular cells (HPA: Colon glandular cells, High). Use adipose tissue as the negative tissue and expect adipocytes to lack detectable staining (HPA: Adipose tissue adipocytes, Not detected); the supplied colon row establishes no verified internal negative cell population, so unstained nonglandular areas are only a local background reference (HPA: Colon glandular cells, High).

Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EIF4A1 in CACO-2, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and an EIF4A1 knockout preparation processed in parallel if available (selected IHC caption: rabbit primary antibody; standard IHC practice). For the colon slide, block endogenous peroxidase and check for endogenous biotin background when using the reported biotin–SABC/DAB detection (selected IHC caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (selected IHC caption: fixative not stated). The reported workflow uses heat-mediated citrate retrieval at pH 6 for 20 minutes in human colon cancer sections; whether retrieval is required in other preparations is unreported (selected IHC caption: citrate retrieval, pH 6, 20 minutes). Frozen sections and IF/ICC are not established as easier by the supplied evidence, although ICC-IF images support cytosolic localization (HPA subcellular: Cytosol, approved); colon staining with the reported biotin–SABC/DAB method warrants checking endogenous biotin and peroxidase background (selected IHC caption: SABC/DAB; standard IHC practice).

HPA tissue IHC evidence for EIF4A1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Placenta	Trophoblastic cells	High	Protein (IHC)	HPA →
Rectum	Glandular cells	High	Protein (IHC)	HPA →
Stomach	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 →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Cerebral cortex	Endothelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced EIF4A1 IHC Tips

Troubleshoot EIF4A1 staining in paraffin sections by checking retrieval, cytoplasmic localisation, controls and cell-specific scoring before interpreting DAB intensity.

How should I retrieve EIF4A1 in paraffin sections when staining is weak?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A03922-2; selected-SKU IHC caption). The selected paraffin-section example used that retrieval before overnight incubation with 1 μg/mL primary antibody at 4°C, so reproduce its retrieval conditions when assessing weak staining (selected-SKU IHC caption). Check that sections remain covered by buffer during heating, then compare matched sections processed together to separate retrieval variation from detection variation (standard IHC practice). If staining remains weak, optimise heating and primary-antibody concentration on adjacent sections while retaining a positive control with cytoplasmic staining (standard IHC practice; HPA: cytoplasmic expression in several tissues).

Could fixation explain weak or uneven EIF4A1 staining?

The selected paraffin-section caption does not state a fixative, and target-specific fixation sensitivity for EIF4A1 is unknown from the supplied evidence (selected-SKU IHC caption). Record each specimen’s fixative and fixation duration before comparing staining, because fixation conditions can change antigen accessibility in paraffin sections (standard IHC practice). Process a control section alongside test sections with the same retrieval and detection steps, and inspect both for patchy staining or tissue damage (standard IHC practice). Do not attribute a fixation effect to EIF4A1 from its cytoplasmic location, modified residues or reported tissue patterns; those records do not test fixation sensitivity (UniProt P60842 localisation and modified residues; HPA tissue IHC).

Which staining compartments are plausible for EIF4A1 in tissue sections?

Evaluate cytoplasmic staining first, including possible perinuclear accentuation, because those locations are reported for EIF4A1 (UniProt P60842 localisation). UniProt also lists cell-membrane localisation and stress-granule recruitment after arsenate or hydrogen peroxide treatment, but the record reports no transmembrane segment (UniProt P60842 localisation and topology). Assess a sharp membranous rim or punctate pattern against adjacent cells and the specimen context before calling it EIF4A1-specific (standard IHC practice; UniProt P60842 localisation). HPA reports cytoplasmic tissue expression, although its subcellular cytosol assignment cautions that the supporting antibodies target proteins from multiple genes (HPA tissue IHC; HPA subcellular).

How can isoforms and epitope placement affect EIF4A1 IHC interpretation?

EIF4A1 has 3 listed isoforms, but the supplied antibody caption does not identify its recognised epitope or establish isoform coverage (UniProt P60842 isoforms; selected-SKU IHC caption). The protein contains an ATP-binding helicase region at residues 63–234 and a C-terminal helicase region at 245–406 (UniProt P60842 domains). Reported modified residues include positions 2, 4, 118 and 158; their effect on this antibody’s staining has not been established (UniProt P60842 modified residues; selected-SKU IHC caption). If staining differs between specimens, check the antibody’s documented immunogen or epitope and use an independent reagent where specificity matters (standard IHC practice).

How could IF help assess an ambiguous EIF4A1 IHC pattern?

Use IF as a separate localisation check when chromogenic tissue staining is ambiguous; EIF4A1 is reported in the cytoplasm and perinuclear region (UniProt P60842 localisation). Multiplex EIF4A1 with a marker for the expected cell type, such as a glandular-cell marker when examining colon sections, and check that the signals occupy the same cells (HPA: high in colon glandular cells; standard IF practice). Choose spectrally separated fluorophores and image an unstained section to identify tissue autofluorescence before interpreting weak signal (standard IF practice). Because EIF4A1 has no transmembrane segment and is reported in cytoplasmic compartments, include permeabilisation when testing intracellular epitopes, with matched controls for background (UniProt P60842 topology and localisation; standard IF practice).

What should I check when EIF4A1 DAB staining appears diffuse?

The selected tissue example used 10% goat serum blocking, a biotinylated secondary antibody and streptavidin-biotin detection with DAB (selected-SKU IHC caption). Check a section without primary antibody and review the peroxidase-block step, because residual endogenous enzyme activity can create chromogenic background (standard IHC practice). In a biotin-based workflow, evaluate endogenous biotin as another potential source of signal, particularly when staining appears outside cells (standard IHC practice; selected-SKU IHC caption). Compare suspect deposits with the expected cytoplasmic distribution, and assess section edges and damaged areas separately before changing the antibody concentration (UniProt P60842 localisation; standard IHC practice).

How should I quantify EIF4A1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and cytoplasmic scoring area before comparing sections, since HPA reports differing staining levels among cell types (HPA tissue IHC; standard IHC practice). For intact cells, report the percentage positive and an H-score based on staining intensity; state the intensity categories and threshold used (standard IHC practice). If counting discrete positive cells, express density per mm² of viable, assessed tissue and exclude necrotic or folded regions consistently (standard IHC practice). Normalise comparisons to the same cell type and tissue area, and include a jointly processed control section because HPA rates its tissue staining evidence as uncertain (standard IHC practice; HPA tissue IHC reliability).

When does an EIF4A1-positive DAB pattern warrant skepticism?

A convincing result should identify stained cells and show predominantly cytoplasmic signal, consistent with the reported tissue profile and cellular localisation (HPA tissue IHC; UniProt P60842 localisation). Recheck isolated nuclear-only staining, strong section-edge colour, necrotic deposits or signal persisting without primary antibody before calling a specimen positive (standard IHC practice; UniProt P60842 localisation). Compare the cell type with the claim: HPA reports high staining in colon glandular cells but no detection in adipocytes, while rating its overall tissue IHC reliability uncertain (HPA tissue IHC). Confirm consequential findings with an independent specificity control and matched processing, especially when the observed compartment or cell population conflicts with the reference pattern (standard IHC practice; HPA tissue IHC reliability).

Boster reagents

Best EIF4A1 / Eukaryotic initiation factor 4A-I IHC Antibodies

The catalog shows EIF4A1 IHC in human and mouse paraffin sections and IF/ICC in human cells (catalog image captions); all four pictured antibodies list human, mouse and rat reactivity (catalog).

Real IHC data

Anti-EIF4A1/2/3 Antibody ®

Cat # A03922-2

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Real IHC data

Anti-EIF4A1 Antibody ® (monoclonal, 3F11)

Cat # M03922-2

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Real IHC data

Anti-EIF4A1 Antibody ®

Cat # A03922-3

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Real IHC data

Anti-EIF4A1 Antibody ® (monoclonal, 11B8)

Cat # M03922-1

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A03922-2 has IHC images from human colon cancer and mouse intestine, while A03922-3 has IHC images from human tonsil and mouse spleen (catalog image captions). M03922-1 has IHC images from human gastric and breast cancers; M03922-2 has images from human placenta, colonic adenocarcinoma and tonsil, plus mouse colon (catalog image captions).

Which to pick: For EIF4A1 tissue IHC, start with A03922-3 for human tonsil or mouse spleen paraffin sections (A03922-3 IHC captions); A03922-2 also detects EIF4A2/3 (catalog title). For IF/ICC, A03922-3 has an A431 cell image and a 2 μg/mL catalog dilution (A03922-3 IF caption; catalog). For human and mouse tissue work, M03922-2 has images in both species and lists rat reactivity, although its pictured sections are paraffin embedded and their fixative is unreported (M03922-2 IHC captions; catalog reactivity).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P60842 (IF4A1_HUMAN, Eukaryotic initiation factor 4A-I).
Human Protein Atlas. EIF4A1 tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. EIF4A1 subcellular location (ICC-IF): Localized to the cytosol. Caution: Based on antibodies targeting proteins from multiple genes..
Human Protein Atlas. EIF4A1 antibody validation summary (2 antibodies).
Novel eIF4A1 inhibitors with anti-tumor activity in lymphoma. Molecular medicine (Cambridge, Mass.) 2022 — PMC9441068.
Aberrant Expression Of PDCD4/eIF4A1 Signal Predicts Postoperative Recurrence For Early-Stage Oral Squamous Cell Carcinoma. Cancer management and research 2019 — PMC6857661.
Expression pattern and prognostic value of key regulators for N7-methylguanosine RNA modification in prostate cancer. Acta biochimica et biophysica Sinica 2023 — PMC10195147.
Identification and Verification of m(7)G Modification Patterns and Characterization of Tumor Microenvironment Infiltration via Multi-Omics Analysis in Clear Cell Renal Cell Carcinoma. Frontiers in immunology 2022 — PMC9113293.
PubMed PMID:8493113 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:16625196 — UniProt-cited evidence.