EPB41L2 / Band 4.1-like protein 2 · IHC design guide

Design Immunohistochemistry for EPB41L2

Plan EPB41L2 chromogenic paraffin IHC with colon glandular cells as a positive control and prostate glandular cells as a negative comparator (HPA tissue IHC). This guide covers cytoplasmic and membranous staining (HPA tissue IHC) and the catalog antibody's 1:100–1:300 IHC dilution (datasheet A03718).

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

Immunohistochemistry protocol sheet for EPB41L2 (IHC for EPB41L2): expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC), antibody A03718, validated IHC image, and IHC protocol steps — Printable **EPB41L2** IHC protocol sheet — expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC), antibody A03718, controls and protocol steps. Open the full EPB41L2 IHC guide →

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EPB41L2 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 membranous tissue staining (HPA tissue IHC)
	Staining pattern	Glandular cells show cytoplasmic and membranous staining (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A03718)
	Positive control ⓘ	Appendix+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	Glandular staining is high or undetected depending on tissue (HPA tissue IHC)
	Regulation	No expression regulator reported (UniProt)
	Isoform / epitope	4 isoforms; no transmembrane segment; epitope unreported (UniProt)

Section 1

Recommended EPB41L2 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet A03718) with a published liver-section IHC protocol (PMC13226250).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A03718)
Fixation	Image fixative and duration unreported (datasheet A03718); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Tris-EDTA pH 9.0 (datasheet A03718); 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-EPB41L2, 1:100-1:300 (datasheet A03718)
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	EPB41L2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in several tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated Tris-EDTA retrieval at pH 9.0 (datasheet A03718); citrate retrieval at pH 6.0 is a published alternative (PMC13226250).

Section 2

What Is the Expected EPB41L2 Staining Pattern?

EPB41L2 is a cytoskeletal protein found in the cytoplasm, cell cortex, and at the cell membrane; it has no transmembrane segment (UniProt O43491). In tissue IHC, expect cytoplasmic and membranous staining in selected cell populations, including appendix glandular cells and bronchial basal cells (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA data; external verification is pending (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic or membranous signal is clear in appendix or colon glandular cells, bronchial basal cells, or bone marrow hematopoietic cells.	This matches the reported tissue compartments and cell populations; HPA records High staining in each example (HPA tissue IHC). Compare signal within the identified cell type, since a tissue-level label does not mean every cell on the section must stain.
Signal is confined to nuclei, with no convincing cytoplasmic or membranous pattern in tissue IHC.	This diverges from the reported tissue IHC pattern (HPA tissue IHC). Check staining specificity and section interpretation before calling it positive. Nucleoplasmic localization is reported in ICC-IF, so a nuclear signal alone cannot establish an artefact (HPA subcellular ICC-IF).
Adipocytes or cardiomyocytes stain as strongly as the selected positive cells.	HPA reports EPB41L2 as Not detected in those cell populations (HPA tissue IHC). Recheck cell identification and controls; cross-reactivity or endogenous detection activity are possible explanations (general IHC practice). A single discordant section does not prove either cause.
Color spreads across cell types and tissue spaces, obscuring cell borders and cytoplasm.	The expected cell-associated pattern cannot be scored reliably (HPA tissue IHC). Diffuse deposit may reflect nonspecific primary-antibody binding, insufficient blocking or washing, or detection background (general IHC practice); inspect the negative control before assigning a cause.
No signal appears in appendix glandular cells or another HPA High population.	The result conflicts with the reported positive population (HPA tissue IHC). First assess section quality, antibody and detection controls, and the identification of the expected cells (general IHC practice). HPA's Supported rating and pending external verification also limit certainty about any individual specimen (HPA tissue IHC).

💡Expected EPB41L2 appearanceCall a positive result when distinct cytoplasmic and membranous chromogenic signal is visible in an HPA High population such as appendix glandular cells; diffuse tissue-wide deposit or equally strong staining in HPA Not detected adipocytes is suspect (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell selection	EPB41L2 is widely expressed (UniProt O43491), but HPA staining varies by cell population: appendix glandular cells are High, thyroid glandular cells are Low, and adipocytes are Not detected (HPA tissue IHC). Select and score the actual cell population, rather than treating a whole organ as uniformly positive or negative.
Compartment and topology	UniProt places EPB41L2 in the cytoskeleton, cell cortex, and at the cell membrane, with no transmembrane segment (UniProt O43491). HPA describes tissue IHC as cytoplasmic and membranous (HPA tissue IHC). Membrane-associated signal therefore fits the record without implying that EPB41L2 spans the membrane.
Antibody validation and isoforms	HPA005730 and HPA006642 each have Supported IHC status; their ICC status is Enhanced (HPA antibodies). UniProt lists four EPB41L2 isoforms (UniProt O43491). These facts do not establish which isoforms either antibody detects, so avoid an isoform-specific interpretation without separate epitope evidence.
What does IF/ICC add?	HPA ICC-IF places EPB41L2 mainly at the plasma membrane, with additional supported nucleoplasmic and cell-junction localization (HPA subcellular ICC-IF). Use that as compartment context for the separate IF/ICC guide; it does not replace the cytoplasmic and membranous tissue IHC pattern or provide an IHC protocol (HPA tissue IHC; HPA subcellular ICC-IF).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A selected HPA High cell population has no visible chromogenic signal.	The expected cell type may be absent from the examined area, or an IHC workflow or detection step may have failed (HPA tissue IHC; general IHC practice).	Confirm the cell population on a counterstained section, then review the catalog antibody's IHC-P instructions and run appropriate positive and detection controls (general IHC practice). Do not infer EPB41L2-specific fixation sensitivity from this result.
Signal appears only in nuclei in a tissue section.	The compartment differs from HPA's cytoplasmic and membranous tissue profile, although nucleoplasmic signal is supported in ICC-IF (HPA tissue IHC; HPA subcellular ICC-IF).	Compare with the expected cytoplasmic and membranous cells on the same section and inspect negative controls (HPA tissue IHC; general IHC practice). Record nuclear-only tissue staining as unresolved until independently checked.
Cells reported as Not detected show prominent staining.	A mismatch with the HPA cell-level pattern may reflect cell misidentification, cross-reactivity, or endogenous detection activity (HPA tissue IHC; general IHC practice).	Verify morphology and review primary-omission and detection controls (general IHC practice). Compare with a documented High population, such as bronchial basal cells, before accepting the signal as EPB41L2 (HPA tissue IHC).
Background masks cell borders or fills tissue spaces.	Nonspecific binding, insufficient blocking or washing, or detection background can obscure a localized IHC pattern (general IHC practice).	Inspect the primary-omission control, blocking and wash steps, and chromogen development (general IHC practice). Score EPB41L2 only where a cell-associated cytoplasmic or membranous pattern can be resolved (HPA tissue IHC).
Two cell populations in one section have different staining intensities.	Cell-level variation is plausible: HPA reports High, Low, and Not detected populations across tissues (HPA tissue IHC).	Identify and score each cell population separately, using the relevant HPA entry as a reference (HPA tissue IHC). Do not average a strong focal signal into a whole-tissue score.
A membrane-rich IF image seems inconsistent with a cytoplasmic tissue IHC section.	ICC-IF reports mainly plasma-membrane localization, while tissue IHC reports cytoplasmic and membranous expression (HPA subcellular ICC-IF; HPA tissue IHC).	Interpret each preparation against its own HPA localization evidence; use the separate IF/ICC guide for IF decisions (HPA subcellular ICC-IF; HPA tissue IHC). Neither image alone establishes an IHC-specific epitope or fixation effect.

Source: UniProt O43491 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing EPB41L2 staining

Immunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used for antigen retrieval. 3,Secondary antibody was diluted at 1:200 (room temperature, 30min).

A03718 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000079819-EPB41L2/tissue/cerebral+cortex#imid_2390880

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000079819-EPB41L2/tissue/colon#imid_2390822

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000079819-EPB41L2/tissue/liver#imid_2390823

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000079819-EPB41L2/tissue/kidney#imid_2390816

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA005730; green = EPB41L2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for EPB41L2 IHC & IF

🧪Run appendix first: glandular cells should stain (HPA: High in appendix glandular cells). Use prostate glandular cells as the tissue negative (HPA: Not detected in prostate glandular cells); on the appendix slide, cells independently verified as EPB41L2-negative should show counterstain and background chromogen only.

Positive control tissue: Appendix (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EPB41L2 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality, and EPB41L2-knockout material as a biological negative. In appendix, block endogenous peroxidase and inspect inflammatory cells for residual chromogen background (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected A03718 paraffin-section caption also leaves the fixative unreported (selected A03718 caption). That caption uses Tris-EDTA at pH 9.0 for retrieval and a 1:200 primary dilution overnight at 4°C, but does not establish whether retrieval is required (selected A03718 caption). Frozen-section performance is unreported; IF/ICC offers a way to assess the reported plasma-membrane and cell-junction localisation, while appendix inflammatory cells warrant attention to endogenous peroxidase in chromogenic IHC (HPA subcellular; standard IHC practice).

HPA tissue IHC evidence for EPB41L2

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium 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
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Bronchus	Basal cells	High	Protein (IHC)	HPA →
Caudate	Glial cells	High	Protein (IHC)	HPA →
Cervix	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 →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Placenta	Decidual cells	Not detected	Protein (IHC)	HPA →
Prostate	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced EPB41L2 IHC Tips

Use documented staining patterns, the catalog antibody’s paraffin-section conditions, and matched controls to troubleshoot EPB41L2 chromogenic IHC.

Which retrieval conditions should I try first for weak EPB41L2 staining?

Use heat-mediated Tris-EDTA at pH 9.0 for antigen retrieval in paraffin sections (datasheet A03718). Start with the catalog antibody at 1:200 after retrieval and incubate at 4°C overnight; compare adjacent sections if heating damages tissue architecture (datasheet A03718; standard IHC practice). Judge improvement in cytoplasmic and membranous staining within intact cells, and check whether apparent gains occur only along torn or lifted section edges (HPA: tissue IHC profile; standard IHC practice). If staining remains weak, test another retrieval buffer or heating duration on adjacent sections while holding antibody and detection conditions constant, then select the condition that preserves morphology and specific staining (standard IHC practice).

Could fixation explain inconsistent EPB41L2 staining between paraffin blocks?

The A03718 paraffin-section caption does not report a fixative, so EPB41L2-specific fixation sensitivity is unknown (datasheet A03718). Record each block’s fixative, fixation duration, processing history, and section age before comparing staining across blocks (standard IHC practice). Hold retrieval at Tris-EDTA pH 9.0 and antibody dilution at 1:200 initially, and include the same positive control section in each run (datasheet A03718; standard IHC practice). If signal differs, compare similarly processed specimens and change one processing variable at a time; tissue-expression patterns and protein topology do not establish a target-specific fixation effect (HPA: tissue IHC profile; UniProt O43491; standard IHC practice).

How should I assess membrane, cytoplasmic, and nuclear EPB41L2 staining?

Assess cell-border and cytoplasmic staining first because these patterns are reported in tissue IHC and fit EPB41L2’s cortex, cytoskeleton, and membrane annotation (HPA: tissue IHC profile; UniProt O43491 localisation). EPB41L2 has no transmembrane segment, so a border pattern need not indicate that the protein crosses the membrane (UniProt O43491 topology). HPA also reports supported nucleoplasmic localisation in cell imaging; evaluate nuclear staining separately rather than automatically calling it artefact (HPA: subcellular localisation). Compare each compartment with a matched negative control and with intact cells away from section edges, then document which cells and compartments contributed to the chromogenic score (standard IHC practice).

Could isoforms or epitope accessibility change the IHC pattern?

EPB41L2 has 4 annotated isoforms and a FERM domain spanning residues 218–499, but the supplied A03718 caption does not map its antibody epitope (UniProt O43491; datasheet A03718). Do not assign a missing tissue signal to one isoform without epitope mapping and independent isoform evidence (standard IHC interpretation). EPB41L2 also carries annotated modified residues, including phosphoserines, yet their effect on this antibody’s staining is unreported (UniProt O43491; datasheet A03718). If staining changes after retrieval, compare adjacent sections under controlled conditions and report the observed compartment and cell type without claiming isoform or modification specificity (standard IHC practice).

How can IF help resolve an ambiguous chromogenic EPB41L2 pattern?

Use the separate IF/ICC guide for the fluorescence workflow; HPA has EPB41L2 cell images from A-431, U-251MG, and U2OS (HPA: subcellular localisation). For a tissue comparison, multiplex EPB41L2 with a marker for the cell population being assessed and include single-stain controls to check channel bleed-through (standard IF practice). Choose spectrally separated fluorophores and inspect an unstained section for autofluorescence before interpreting dim border signal (standard IF practice). Because EPB41L2 has no transmembrane segment and is annotated at the cortex, cytoskeleton, and membrane, assess whether the antibody epitope is accessible from the cytoplasmic side and adjust permeabilisation accordingly (UniProt O43491 topology and localisation; standard IF practice).

What should I change when EPB41L2 chromogenic staining is diffuse?

First inspect a no-primary control to separate antibody-associated staining from detection-system background, and compare it with a section stained using the catalog antibody (standard IHC practice). Block endogenous peroxidase for enzyme-based chromogenic detection, then check whether residual pigment or precipitated chromogen resembles the putative EPB41L2 signal (standard IHC practice). If background follows tissue edges or folds, improve section handling and washing before increasing interpretation thresholds (standard IHC practice). If intact cells remain diffusely stained, titrate around the caption’s 1:200 primary dilution while keeping retrieval at Tris-EDTA pH 9.0 and comparing the same positive and negative controls (datasheet A03718; standard IHC practice).

How should I score EPB41L2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and score membranous and cytoplasmic staining separately, since both patterns occur in HPA tissue IHC (HPA: tissue IHC profile; standard IHC practice). An H-score can sum the percentage of cells at each 0–3 intensity level, giving a 0–300 range; report the scoring rule consistently across sections (standard IHC practice). Alternatively report percentage of positive cells or positive-cell density per mm², using the same threshold and sampled tissue area for every specimen (standard IHC practice). Normalise to eligible intact cells or viable tissue area, and exclude folds, necrosis, and edge artefacts before comparing groups (standard IHC practice).

How do I distinguish convincing EPB41L2 signal from artefact?

Look for reproducible staining in intact cell borders or cytoplasm, consistent with the reported tissue pattern and EPB41L2 localisation (HPA: tissue IHC profile; UniProt O43491 localisation). HPA reports high staining in appendix glandular cells and no detection in adipose-tissue adipocytes, which can inform control selection without guaranteeing every specimen’s result (HPA: appendix glandular cells, High; adipose-tissue adipocytes, Not detected). Treat signal confined to edges, necrotic areas, or no-primary controls as suspect, and check endogenous enzyme activity in chromogenic assays (standard IHC practice). Nuclear signal warrants separate review because HPA supports nucleoplasmic localisation in cell imaging; confirm its cellular context and reproducibility before interpreting it (HPA: subcellular localisation; standard IHC practice).

Boster reagents

Best EPB41L2 / Band 4.1-like protein 2 IHC Antibodies

Both anti-EPB41L2 antibodies list IHC and human/mouse reactivity (catalog: A03718, A03718-1); only A03718 includes a paraffin-section IHC image (A03718 image caption).

Real IHC data Immunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used for antigen retrieval. 3,Secondary antibody was diluted at 1:200 (room temperature, 30min).

Anti-4.1G Antibody

Cat # A03718

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A03718 lists human/mouse reactivity and IHC/IF applications (catalog: A03718 applications/reactivity); its IHC image shows paraffin-embedded human tonsil (A03718 image caption). A03718-1 lists human/mouse/rat reactivity and IHC, with no IHC or IF image supplied (catalog: A03718-1 applications/reactivity/images).

Which to pick: For tissue IHC, choose A03718 when a demonstrated paraffin-section workflow matters: its human tonsil image reports 1:200 antibody dilution and Tris-EDTA pH 9.0 retrieval; the fixative is unreported (A03718 image caption). For IF/ICC, A03718 is the only IF-listed option, though neither an IF image nor ICC validation is supplied (catalog: A03718 applications/images). For rat IHC, A03718-1 lists rat reactivity but has no example image; both antibodies have rabbit hosts and unspecified clones, so clonality does not distinguish them (catalog: A03718 and A03718-1 host/clone; A03718-1 reactivity/applications/images).

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

References

UniProt Consortium. UniProt entry O43491 (E41L2_HUMAN, Band 4.1-like protein 2).
Human Protein Atlas. EPB41L2 tissue IHC expression (reliability: Supported).
Human Protein Atlas. EPB41L2 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the nucleoplasm and cell junctions..
Human Protein Atlas. EPB41L2 antibody validation summary (2 antibodies).
Unveiling novel macrophage-specific biomarkers in MASH through single-cell sequencing for diagnostic modeling. Journal of lipid research 2026 — PMC13226250.
EPB41L family serves as a prognostic biomarker for kidney renal clear cell carcinoma. Cell adhesion & migration 2026 — PMC12885433.
Changes in cortical cytoskeletal and extracellular matrix gene expression in prostate cancer are related to oncogenic ERG deregulation. BMC cancer 2010 — PMC2955608.
Delayed astrocyte development impairs Sema6a-Plxna2/4-mediated astrocyte-neuron crosstalk and causes depressive-like behavior. Nature communications 2026 — PMC13494039.
PubMed PMID:9598318 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:17974005 — UniProt-cited evidence.