KPNA4 / Importin subunit alpha-3 · IHC design guide

Design Immunohistochemistry for KPNA4

Plan chromogenic KPNA4 IHC around the mainly nuclear tissue pattern (HPA tissue IHC). The guide uses paraffin-section examples from human esophageal cancer and rat heart with catalog antibody A05188-1 at 1:200 (datasheet: IHC captions).

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

Immunohistochemistry protocol sheet for KPNA4 (IHC for KPNA4): expected localisation Mainly nuclear in tissues (HPA tissue IHC), antibody A05188-1, validated IHC image, and IHC protocol steps — Printable **KPNA4** IHC protocol sheet — expected localisation Mainly nuclear in tissues (HPA tissue IHC), antibody A05188-1, controls and protocol steps. Open the full KPNA4 IHC guide →

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KPNA4 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	Mainly nuclear in tissues (HPA tissue IHC)
	Staining pattern	Mainly nuclear staining across most tissues (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

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 A05188-1)
	Caveat	Colon glands stain high despite low reported colon expression (HPA tissue IHC; UniProt)
	Regulation	High in muscle; low in kidney (UniProt)
	Isoform / epitope	No listed isoforms; intracellular chain spans aa 2–521 (UniProt)

Section 1

Recommended KPNA4 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with published KPNA4 IHC methods for spinal cord, pancreas, hepatocellular carcinoma, and frontal cortex (PMC12325296; PMC9002131; PMC7950262; PMC6158706).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat heart tissue; fixative not specified (datasheet A05188-1)
Fixation	Image fixative and duration unreported (datasheet A05188-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-KPNA4, 1:50-1:200 (datasheet A05188-1)
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	KPNA4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Mainly nuclear expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); citrate retrieval was used for the neural tissues (PMC12325296; PMC6158706).

Section 2

What Is the Expected KPNA4 Staining Pattern?

KPNA4 staining in paraffin sections should be mainly nuclear across most tissues, including glandular and respiratory epithelial cells with reported high staining (HPA tissue IHC: Enhanced reliability; medium consistency with RNA). UniProt also places KPNA4 in the cytoplasm and nucleus and reports no transmembrane segment (UniProt O00629: subcellular location and topology). Interpret staining by compartment and cell type together.

What am I looking at on my slide?
Distinct nuclear chromogen in bronchial respiratory epithelium or adrenal glandular cells, with visible unstained tissue detail.	This fits the reported high staining in those cells and the mainly nuclear tissue profile (HPA tissue IHC: High; mainly nuclear). Compare intensity within the same run rather than treating every stained cell as equally informative (standard IHC practice).
Strong, widespread cytoplasmic staining with little or no nuclear signal in a tissue expected to stain strongly.	Question the IHC result because it departs from HPA’s mainly nuclear tissue pattern (HPA tissue IHC). Some cytoplasmic KPNA4 remains biologically plausible (UniProt O00629: cytoplasm; nucleus), so compartment alone cannot prove an artefact. Check controls and staining conditions (standard IHC practice).
Prominent staining in cells outside the reported positive population, while the expected cells are weak.	For example, a slide that misses bronchial respiratory epithelium does not match its reported high staining (HPA tissue IHC: bronchus). Unexpected staining may reflect cross-reactivity or endogenous detection activity; use controls before assigning either cause (standard IHC practice).
Uniform chromogen haze across nuclei, cytoplasm and surrounding tissue, obscuring cell boundaries.	This is difficult to score as compartment-specific KPNA4 because the expected tissue pattern is mainly nuclear (HPA tissue IHC). Diffuse background can arise from detection or blocking conditions; inspect the no-primary control and adjust the general IHC workflow (standard IHC practice).
No convincing nuclear signal in a control section containing bronchial respiratory epithelium or adrenal glandular cells.	Both populations have reported high KPNA4 staining (HPA tissue IHC: bronchus; adrenal gland). First check that the expected cells are present, then review antibody, retrieval and detection controls (standard IHC practice). A failed run cannot establish absence of KPNA4.

💡Expected KPNA4 appearanceCall positive when identifiable cells show predominantly nuclear chromogen, especially in reported high-staining glandular or respiratory epithelial populations (HPA tissue IHC); broad, compartment-independent haze is suspect background (standard IHC practice).

How each factor affects the staining
Tissue and cell choice	HPA reports high staining in several populations, including adrenal and appendix glandular cells, bronchial respiratory epithelium, and neurons in caudate and cortex (HPA tissue IHC). Nasopharyngeal respiratory epithelium and salivary glandular cells are listed as low, so matched cell identity matters more than the organ name alone (HPA tissue IHC).
Compartment evidence across applications	Tissue IHC is mainly nuclear (HPA tissue IHC). ICC-IF places KPNA4 mainly in the nucleoplasm, with additional nuclear membrane localization (HPA subcellular: enhanced nucleoplasm; approved nuclear membrane). Those IF observations support interpretation of localization; they do not specify an IHC-P protocol.
Antibody validation	HPA lists HPA045500 and CAB034336 as IHC Enhanced, meaning their pattern has support from independent antibodies or orthogonal data; HPA043154 is IHC Supported (HPA antibodies). The overall tissue reliability is Enhanced but has only medium consistency with RNA, so a single discrepant slide warrants controls (HPA tissue IHC).
Expression evidence differs by source	UniProt describes KPNA4 as barely detectable in colon, whereas HPA reports high staining in colon glandular cells (UniProt O00629: tissue specificity; HPA tissue IHC: colon glandular cells, High). Treat colon as a source-dependent example, and prefer an unambiguous HPA high-staining control when assessing the HPA IHC pattern.
Molecular context	KPNA4 has no transmembrane segment or signal peptide and functions in nuclear protein import (UniProt O00629: topology; processing; function). These facts support interpreting intracellular staining, but do not establish that any particular retrieval or fixation condition changes the IHC signal.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Positive control has no nuclear chromogen.	The section may lack the expected cell population, or the staining run may have failed (standard IHC practice).	Confirm bronchial respiratory epithelium or adrenal glandular cells are present (HPA tissue IHC: High); review the antibody, retrieval and detection steps with run controls (standard IHC practice).
Signal appears only after very strong chromogen development.	Weak specific signal and accumulated background can become hard to distinguish (standard IHC practice).	Compare the positive tissue and no-primary control at the same development time, then adjust detection conditions if needed (standard IHC practice). Do not score haze as KPNA4 localization.
Cytoplasm dominates while nuclei remain pale.	The pattern differs from mainly nuclear tissue IHC, although cytoplasmic localization is recorded by UniProt (HPA tissue IHC; UniProt O00629: location).	Verify cell identity and compare with a reported high-staining control; review retrieval and antibody conditions without assuming a KPNA4-specific fixation effect (standard IHC practice).
Chromogen appears in the no-primary control.	Background may come from the detection system, including endogenous activity when the detection chemistry permits it (standard IHC practice).	Address the relevant endogenous activity and review blocking and detection reagents before interpreting the test section (standard IHC practice).
Colon glandular staining differs from the expected control result.	The sources disagree: HPA reports high glandular staining, while UniProt calls colon expression barely detectable (HPA tissue IHC: colon; UniProt O00629: tissue specificity).	Record the discrepancy and assess the same run with another HPA high-staining tissue, such as bronchus or adrenal gland (HPA tissue IHC: High); avoid using colon alone to judge assay success.
IF/ICC shows nuclear rim signal as well as nucleoplasmic signal.	HPA records additional nuclear membrane localization in ICC-IF (HPA subcellular: nuclear membrane, approved).	Interpret this alongside the enhanced nucleoplasmic location (HPA subcellular). Use the separate IF/ICC guide for its staining workflow; this IHC section supplies no IF protocol.

Source: UniProt O00629 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing KPNA4 staining

Immunohistochemistry of paraffin-embedded rat heart using KPNA4 Antibody at dilution of 1:200 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

A05188-1 · IHCImmunohistochemistry of paraffin-embedded rat heart using KPNA4 Antibody at dilution of 1:200 .Perform microwave antigen retrieval with 10 m

Immunohistochemistry of paraffin-embedded human esophageal cancer using KPNA4 Antibody at dilution of 1:200 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

A05188-1 · IHCImmunohistochemistry of paraffin-embedded human esophageal cancer using KPNA4 Antibody at dilution of 1:200 .Perform microwave antigen retri

A05188-1 · IFImmunofluorescence analysis of U2OS cells using KPNA4 antibody . Blue: DAPI for nuclear staining.

A05188 · IFImmunocytochemistry of KPNA4 in HeLa cells with KPNA4 antibody at 2.5 μg/mL.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000186432-KPNA4/tissue/cerebral+cortex#imid_12987595

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000186432-KPNA4/tissue/colon#imid_12987521

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000186432-KPNA4/tissue/liver#imid_12987522

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000186432-KPNA4/tissue/kidney#imid_12987531

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045500; green = KPNA4, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for KPNA4 IHC & IF

🧪Run adrenal gland first and assess staining in its glandular cells (HPA: High in adrenal gland glandular cells). HPA detects KPNA4 in all 45 scored tissues, so use no-primary and isotype controls in place of a negative tissue; non-glandular cells on the positive slide should be assessed for background staining without assuming they are KPNA4-negative (HPA: no negative tissue listed).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: KPNA4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show KPNA4 in A-431, PC-3, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 2 (10x), U2OS, scrambled (10x), NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control; a nonimmune isotype control matched to the catalog antibody’s host species and clonality; and, if available, a KPNA4 knockout or immunizing-peptide block as a biological specificity control. Quench endogenous peroxidase for chromogenic detection and check the adrenal section for pigment that could be mistaken for chromogen (HPA: adrenal gland is a High positive tissue).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative for the paraffin-section rat-heart image is unreported (selected A05188-1 IHC caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, before staining at 1:200, but does not establish whether retrieval is required (selected A05188-1 IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; HPA ICC-IF images show mainly nucleoplasmic localization with additional nuclear-membrane localization, which can guide assessment of IF signal (HPA: subcellular summary).

HPA tissue IHC evidence for KPNA4

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Caudate	Neuronal cells	High	Protein (IHC)	HPA →
Cerebellum	Bergmann glia - nucleus	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: KPNA4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced KPNA4 IHC Tips

Troubleshoot KPNA4 chromogenic IHC by checking nuclear staining, section processing, and controls before comparing signal across tissues (HPA tissue IHC: mainly nuclear).

What retrieval should I try when KPNA4 nuclear staining is weak?

Start with Tris-EDTA, pH 9.0, heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting: nuclear antigen). Cool sections consistently before staining and compare matched sections with identical detection conditions (standard IHC practice). If nuclear signal remains weak, test the catalog antibody's illustrated microwave retrieval with 10 mM PBS, pH 7.2, alongside the page setting (A05188-1 image caption). The illustrated rat heart section used a 1:200 antibody dilution, which provides a documented comparison point for that specimen (A05188-1 image caption). Judge either condition by nuclear signal, background, and preserved cell boundaries rather than staining intensity alone (HPA tissue IHC: mainly nuclear; standard IHC practice).

Could fixation explain faint or uneven KPNA4 staining?

KPNA4-specific fixation sensitivity is unknown because no target-specific fixation evidence is supplied for this IHC workflow (supplied evidence: fixation not stated). The catalog image identifies a paraffin-embedded rat heart section but does not state its fixative or fixation duration (A05188-1 image caption). Record fixative, fixation duration, processing schedule, and section age for every specimen before attributing differences to KPNA4 abundance (standard IHC practice). Compare sections processed together, using the same retrieval, 1:200 starting dilution where appropriate, and detection settings (A05188-1 image caption; standard IHC practice). If signal varies within a section, inspect morphology and staining gradients before changing antibody concentration (standard IHC practice).

Should KPNA4 stain nuclei, cytoplasm, or both in tissue sections?

Expect predominantly nuclear staining in tissue IHC, while allowing some cytoplasmic signal because KPNA4 is annotated in both compartments (HPA tissue IHC: mainly nuclear; UniProt O00629: cytoplasm and nucleus). The nucleoplasm is the principal location in subcellular imaging, with additional nuclear membrane localisation reported (HPA subcellular: nucleoplasm enhanced; nuclear membrane approved). Score nuclear and cytoplasmic staining separately instead of combining them into one intensity value (standard IHC practice). Use a nuclear counterstain to confirm that chromogen lies within intact nuclei, especially in crowded regions (standard IHC practice). Predominantly extracellular or uniformly diffuse staining warrants a background and morphology check before biological interpretation (UniProt O00629: cytoplasm and nucleus; standard IHC practice).

How should I investigate staining that depends strongly on retrieval conditions?

KPNA4 has no listed isoforms, and its annotated chain spans residues 2–521 (UniProt O00629: isoforms and processing). Its importin-beta-binding domain spans residues 2–58, with N-terminal acetylation at residue 2 and phosphorylation at residue 60 (UniProt O00629: domains and modified residues). The antibody epitope is unspecified in the supplied evidence, so these annotations do not establish a target-specific retrieval response (supplied evidence: epitope not stated). Compare matched sections across the documented retrieval conditions while holding antibody dilution and detection constant (page retrieval setting; A05188-1 image caption; standard IHC practice). If a condition increases diffuse signal along with nuclear signal, assess specificity with appropriate controls before scoring (standard IHC practice).

How can IF help investigate an uncertain KPNA4 IHC pattern?

Use IF/ICC as a separate localisation check: KPNA4 is mainly nucleoplasmic in subcellular images, with additional nuclear membrane localisation (HPA subcellular: nucleoplasm enhanced; nuclear membrane approved). In multiplex imaging, pair KPNA4 with a validated marker for the expected cell population and a nuclear stain, then inspect each channel independently (standard IF practice). Select fluorophores in channels with low specimen autofluorescence and include unstained and single-label controls to assess bleed-through (standard IF practice). KPNA4 has no transmembrane segment and is annotated in the nucleus and cytoplasm; permeabilise fixed cells to access intracellular epitopes, while checking the antibody's stated epitope if available (UniProt O00629: topology and subcellular location; standard IF practice). Do not transfer the paraffin-section retrieval conditions directly to IF/ICC (A05188-1 image caption; standard IF practice).

What should I check when KPNA4 chromogenic staining looks diffuse?

First compare a no-primary control with the stained section to identify signal from detection reagents or endogenous tissue activity (standard IHC practice). Apply an appropriate endogenous peroxidase block before peroxidase-based chromogenic detection, and keep blocking and wash conditions consistent across sections (standard IHC practice). Check whether diffuse chromogen increases with stronger retrieval or longer development, then adjust one variable at a time (standard IHC practice). KPNA4 lacks a transmembrane segment and is annotated in the nucleus and cytoplasm, so broad extracellular deposition is difficult to reconcile with its reported localisation (UniProt O00629: topology and subcellular location). Reassess staining in intact cells with the same nuclear counterstain and exposure to chromogen before calling it positive (HPA tissue IHC: mainly nuclear; standard IHC practice).

How should I quantify KPNA4 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define regions and cell populations before scoring, then record nuclear positivity separately from cytoplasmic staining (HPA tissue IHC: mainly nuclear; standard IHC practice). For a cell-based measure, report percent positive cells and an H-score from 0–300 using intensity categories 0–3 (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of evaluable tissue and exclude folds, necrosis, and section edges (standard IHC practice). Normalise counts to the number of intact cells or evaluable tissue area, and compare sections stained in the same run (standard IHC practice). Document cell composition because the HPA tissue profile varies by cell type and has medium consistency with RNA expression (HPA tissue IHC: profile and reliability description).

How can I distinguish true KPNA4 signal from staining artefact?

A credible pattern includes staining in intact cell nuclei, consistent with the predominantly nuclear tissue profile (HPA tissue IHC: mainly nuclear). Compare cell types within the section rather than treating all brown deposits as equivalent; the HPA profile reports high staining in bronchial respiratory epithelial cells and low staining in lymph-node germinal center cells (HPA tissue IHC: positive and low lists). Examine section edges, folds, and necrotic areas for staining that tracks tissue damage rather than cellular boundaries (standard IHC practice). A positive no-primary control suggests detection-system background, including endogenous enzyme activity when peroxidase detection is used (standard IHC practice). Treat predominantly extracellular staining or a pattern without intact nuclei cautiously, and confirm it with controls and matched sections (UniProt O00629: subcellular location; standard IHC practice).

Boster reagents

Best KPNA4 / Importin subunit alpha-3 IHC Antibodies

Validated anti-KPNA4 options show paraffin-section IHC in rat heart and human esophageal cancer (A05188-1 IHC captions), and IF/ICC in HeLa cells (A05188 image captions).

Real IHC data Immunohistochemistry of paraffin-embedded rat heart using KPNA4 Antibody at dilution of 1:200 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

Anti-Importin subunit alpha-3 KPNA4 Antibody

Cat # A05188-1

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Real IF data Immunocytochemistry of KPNA4 in HeLa cells with KPNA4 antibody at 2.5 μg/mL.

Anti-Importin subunit alpha-3 KPNA4 Antibody

Cat # A05188

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A05188-1 has IHC images from paraffin-embedded rat heart and human esophageal cancer, plus an IF image from U2OS cells (A05188-1 image captions). A05188 has ICC and IF images from HeLa cells and no listed IHC application (A05188 image captions; catalog applications).

Which to pick: Choose A05188-1 for tissue IHC: its paraffin-section captions show 1:200 antibody dilution and microwave retrieval in 10 mM PBS at pH 7.2; the fixative is unreported (A05188-1 IHC captions). Choose A05188 for IF/ICC, supported by HeLa images at 20 μg/mL for IF and 2.5 μg/mL for ICC (A05188 image captions). Both are rabbit antibodies with no clone specified and list Human, Mouse, and Rat reactivity; only A05188-1 lists IHC among its applications (catalog host, clone, reactivity, and applications).

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

References

UniProt Consortium. UniProt entry O00629 (IMA3_HUMAN, Importin subunit alpha-3).
Human Protein Atlas. KPNA4 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. KPNA4 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear membrane..
Human Protein Atlas. KPNA4 antibody validation summary (3 antibodies).
Accumulation of TDP-43 causes karyopherin-α4 pathology that characterises amyotrophic lateral sclerosis. Frontiers in neuroscience 2025 — PMC12325296.
Systematic Characterization of the Clinical Relevance of KPNA4 in Pancreatic Ductal Adenocarcinoma. Frontiers in oncology 2022 — PMC9002131.
Value of KPNA4 as a diagnostic and prognostic biomarker for hepatocellular carcinoma. Aging 2021 — PMC7950262.
A feedback loop between dipeptide-repeat protein, TDP-43 and karyopherin-α mediates C9orf72-related neurodegeneration. Brain : a journal of neurology 2018 — PMC6158706.
PubMed PMID:9168958 — UniProt-cited evidence.
PubMed PMID:9395085 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.