This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
EFEMP2 / EGF-containing fibulin-like extracellular matrix protein 2 · IHC design guide
Design Immunohistochemistry for EFEMP2
Plan chromogenic EFEMP2 IHC around cytoplasmic staining and extracellular deposits (HPA tissue IHC). This guide covers fixation, detection and scoring, with an IHC antibody dilution range of 1:50–1:200 (datasheet: A05653-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EFEMP2 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 | Observed cytoplasm and extracellular deposits (HPA tissue IHC) | |
| Staining pattern | Several tissues show cytoplasmic staining and extracellular deposits (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Extracellular deposits can complicate cell-level scoring (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms listed; no cytoplasmic domain; mature chain 24–443 (UniProt) |
Section 1
Recommended EFEMP2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is presented alongside four published EFEMP2 IHC protocols from glioma, breast, bladder, and lung studies (PMC13509514; PMC6826198; PMC6775297; PMC7034775).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A05653-1) |
| Fixation | Image fixative and duration unreported (datasheet A05653-1); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-EFEMP2, 1:50-1:200 (datasheet A05653-1) |
| 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 | EFEMP2-positive staining in preleptotene spermatocytes of testis (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues. Additional extracellular deposits. No signal in the no-primary control. |
💡Decision noteStart with the page’s citrate pH 6.0 retrieval at 95–98 °C for 20 min; published citrate conditions offer tissue-specific starting points (page retrieval; PMC6826198; PMC6775297).
Section 2
What Is the Expected EFEMP2 Staining Pattern?
EFEMP2 is a secreted extracellular matrix and basement membrane protein that localizes to microfibrils around elastin cores and has no transmembrane segment (UniProt O95967). In paraffin-section IHC, expect extracellular deposits alongside the cytoplasmic staining reported across several tissues; preleptotene spermatocytes in testis show medium staining (HPA tissue IHC). HPA rates the tissue staining Approved and cautions that secretion complicates comparisons between RNA and protein location (HPA tissue IHC).
| Extracellular deposits with cytoplasmic staining; medium staining in testis preleptotene spermatocytes (HPA tissue IHC). | This fits HPA’s observed tissue profile (HPA tissue IHC). Extracellular signal is also consistent with EFEMP2 secretion and matrix localization (UniProt O95967). Judge the testis cell signal at its reported medium level; a darker reaction is not required to recognize that pattern (HPA tissue IHC). |
| Signal is confined to nuclei, with no extracellular deposits or cytoplasmic staining. | A nuclear-only pattern does not match the reported cytoplasmic and extracellular IHC profile (HPA tissue IHC) or the annotated secreted matrix location (UniProt O95967). Treat it as suspect and check the detection controls before assigning it to EFEMP2; location alone cannot identify the source of a false signal (general IHC practice). |
| Strong staining appears in an HPA-listed cell type scored Not detected, such as adipocytes in adipose tissue (HPA tissue IHC). | This conflicts with that cell type’s reported IHC result (HPA tissue IHC). Consider antibody cross-reactivity or endogenous detection activity and compare with a no-primary control (general IHC practice). Assess the named cell type rather than treating an entire tissue as negative: secreted protein can reside outside its producing cells (UniProt O95967). |
| Broad, nearly uniform color obscures cells and extracellular structures. | The slide cannot reliably distinguish HPA’s cytoplasmic expression from its additional extracellular deposits (HPA tissue IHC). Diffuse color may reflect nonspecific antibody binding or detection background; inspect a no-primary control and the spatial pattern before scoring (general IHC practice). Uniform background by itself is not evidence of EFEMP2 distribution (general IHC practice). |
| Preleptotene spermatocytes in testis have no visible signal. | HPA reports medium staining in these cells, so a blank result calls for a run check (HPA tissue IHC). Confirm that the scored cells are present, then review staining and detection controls (general IHC practice). One negative slide cannot establish that EFEMP2 is absent from the sample, particularly for a secreted protein (UniProt O95967). |
💡Expected EFEMP2 appearanceA credible positive shows medium cytoplasmic staining in testis preleptotene spermatocytes, with extracellular deposits fitting the broader tissue profile; nuclear-only color or strong signal in a cell type HPA scores Not detected is suspect (HPA tissue IHC; UniProt O95967).
| Secreted protein and tissue interpretation (UniProt O95967). | EFEMP2 has a signal peptide, lacks a transmembrane segment, and localizes to extracellular matrix and basement membrane (UniProt O95967). Deposited protein can therefore appear apart from its producing cell; HPA specifically cautions that tissue RNA and protein locations may differ for secreted variants (HPA tissue IHC). |
| Strength of the IHC reference pattern (HPA tissue IHC). | HPA calls the tissue profile Approved and reports medium staining in testis preleptotene spermatocytes (HPA tissue IHC). Its listed antibody, HPA023270, is IHC Approved; the payload does not report Enhanced IHC validation or an ICC result for it (HPA antibodies). Use the profile as a reference pattern, not a guarantee for every section (HPA tissue IHC). |
| Processing and matrix association (UniProt O95967). | UniProt annotates a signal peptide at residues 1–23 and a mature chain at 24–443, plus glycosylation sites at 198 and 394 (UniProt O95967). It also places EFEMP2 on microfibrils surrounding elastin cores (UniProt O95967). These facts support interpreting extracellular deposits; they do not establish an antibody epitope or an EFEMP2-specific retrieval condition. |
| Can the IHC result establish an IF/ICC pattern? | No cell-line ICC-IF images or main intracellular location are supplied; HPA’s subcellular summary says Secreted (HPA subcellular ICC-IF). The listed antibody has no ICC validation status in the payload (HPA antibodies). Use the separate IF/ICC guide for that application; do not transfer IHC staining expectations into an IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis preleptotene spermatocytes are blank despite the reported medium IHC signal (HPA tissue IHC). | The relevant cells may be missing from the scored area, or the staining run may have failed (general IHC practice). | Locate the preleptotene spermatocytes on the section and check the run’s positive and detection controls before interpreting the blank result (general IHC practice). |
| Extracellular deposits are present, but little signal is assigned to nearby cells. | EFEMP2 is secreted and deposited in extracellular matrix; protein location need not track the producing cell (UniProt O95967; HPA tissue IHC). | Score extracellular and cytoplasmic staining separately, and record the cell type assessed; do not infer cellular expression solely from adjacent deposits (general IHC practice). |
| A cell type HPA scores Not detected shows strong color, such as adipocytes in adipose tissue (HPA tissue IHC). | Cross-reactivity or endogenous detection activity could account for discordant staining (general IHC practice). | Compare the same cell type with a no-primary control and inspect whether the color follows cells or a nonspecific background pattern (general IHC practice). |
| Color is diffuse across the section and obscures the reported cytoplasmic and extracellular pattern (HPA tissue IHC). | Nonspecific binding, insufficient blocking, or detection background may reduce pattern contrast (general IHC practice). | Review no-primary control staining and the run’s blocking, washing, and detection conditions; reassess localization only when tissue detail is visible (general IHC practice). |
| The stain is nuclear-only. | That location conflicts with the HPA tissue profile and UniProt’s secreted extracellular localization (HPA tissue IHC; UniProt O95967). | Check counterstain and no-primary control appearance, then verify whether any cytoplasmic or extracellular signal remains before scoring EFEMP2 (general IHC practice). |
| A proposed retrieval change is claimed to fix EFEMP2 staining. | The supplied HPA and UniProt records give no EFEMP2-specific fixation sensitivity or retrieval comparison. | Treat retrieval as a general IHC optimization question and compare controlled sections if needed; do not attribute a change in staining to a documented EFEMP2 fixation effect. |
Experiment evidence
IHC & IF images showing EFEMP2 staining
A05653-1 · IHCImmunohistochemistry (IHC) analyzes of Fibulin-4 (D123) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000172638-EFEMP2/tissue/cerebral+cortex#imid_6472080
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000172638-EFEMP2/tissue/colon#imid_6469350
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000172638-EFEMP2/tissue/liver#imid_6469351
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000172638-EFEMP2/tissue/kidney#imid_6469344
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EFEMP2 IHC & IF
🧪Run testis first and assess preleptotene spermatocytes for staining (HPA: Medium in preleptotene spermatocytes); use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes). On the testis slide, use cells without specific chromogen but with intact counterstain as internal negatives; do not assume every other cell type is negative, since EFEMP2 is extracellular (UniProt O95967 extracellular matrix).
Positive control tissue: Testis (Preleptotene spermatocytes, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for EFEMP2; derive a cell-line control from the positive tissue's cell type (Preleptotene spermatocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control; a host species- and immunoglobulin class-matched isotype control suited to the primary antibody’s clonality; and an EFEMP2 knockout specimen or validated peptide-block control (standard IHC practice). Quench endogenous peroxidase for chromogenic testis IHC, and check autofluorescence with an unstained or secondary-only section if using IF (standard IHC/IF practice).
⚠️Feasibility: The selected A05653-1 caption documents paraffin-section IHC at 1:50 but does not report a fixative; a target-specific fixation window, fixation effect, and retrieval dependence are unreported (selected tissue-IHC caption). Whether frozen sections or IF are easier is unestablished; HPA lists no ICC-IF image-bearing cell lines (HPA subcellular). In testis, extracellular matrix signal adjacent to preleptotene spermatocytes could be mistaken for cellular staining, so score extracellular and cell-associated signal separately (UniProt O95967 extracellular matrix; HPA: Medium in preleptotene spermatocytes).
HPA tissue IHC evidence for EFEMP2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 |
|---|---|---|---|---|
| Testis | Preleptotene spermatocytes | Medium | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EFEMP2 IHC Tips
Troubleshoot EFEMP2 staining by checking retrieval, compartment, and controls before interpreting chromogenic signal in paraffin sections.
What retrieval should I start with for EFEMP2 in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). Let sections cool in buffer, then compare retrieved and unretrieved serial sections using the same antibody concentration and detection conditions (standard IHC practice). If staining remains weak, vary heating time in small steps while monitoring tissue integrity and background; a different buffer or pH is a fallback requiring validation (standard IHC practice). Record the retrieval conditions for every comparison, because heat can alter epitope accessibility in paraffin sections (standard IHC practice).
Could fixation explain weak EFEMP2 staining?
The selected antibody image shows paraffin-embedded human breast carcinoma at 1:50, but its caption does not report the fixative (caption: A05653-1). Target-specific sensitivity to fixation is therefore unknown; neither the tissue staining profile nor EFEMP2 topology establishes it (caption: A05653-1; HPA tissue IHC; UniProt O95967 topology). If fixation records are available, compare sections with documented processing while holding retrieval, antibody dilution, and detection constant (standard IHC practice). Examine tissue preservation alongside signal, and use a control section processed with each batch so a processing difference is not mistaken for a change in EFEMP2 abundance (standard IHC practice).
Should EFEMP2 appear in cells or extracellular matrix?
Prioritise extracellular and basement-membrane-associated staining: EFEMP2 is secreted, lacks a transmembrane segment, and localises to matrix microfibrils surrounding elastin cores (UniProt O95967 localisation and topology). Cytoplasmic staining can also occur in tissue IHC, and additional extracellular deposits are reported (HPA tissue IHC). Compare the distribution with tissue architecture rather than assigning every stained cell as the source of deposited protein (UniProt O95967 localisation; standard IHC interpretation). If signal appears exclusively nuclear, review the counterstain, antibody-independent detection control, and serial sections before accepting it as EFEMP2 (UniProt O95967 localisation; standard IHC practice).
How could epitope location affect EFEMP2 IHC?
Check the antibody's immunogen or epitope documentation before interpreting regional staining; the supplied caption identifies the reagent but does not map its epitope (caption: A05653-1). EFEMP2 has a signal peptide at residues 1–23 and a mature chain spanning 24–443, so an epitope confined to the cleaved signal peptide would not represent deposited mature protein (UniProt O95967 processing). The record lists 0 annotated isoforms and glycosylation sites at residues 198 and 394; these annotations do not establish antibody accessibility in paraffin sections (UniProt O95967 isoforms and glycosylation). Compare antibodies against documented, distinct epitopes on serial sections if epitope masking remains a concern (standard IHC practice).
How can I check EFEMP2 localisation by multiplex IF?
Use IF as a separate validation experiment alongside the chromogenic paraffin-section result, whose selected antibody caption reports 1:50 but no IF conditions (caption: A05653-1). In a vascular specimen, pair EFEMP2 with an α-smooth-muscle-actin marker to relate matrix deposits to smooth muscle cells involved in elastic-fiber architecture (UniProt O95967 function; standard IF practice). Choose fluorophores after checking tissue autofluorescence and use single-stain controls to assess spectral bleed-through (standard IF practice). For an extracellular mature-protein epitope, begin without detergent permeabilisation; permeabilise matched sections only when testing an intracellular epitope or secretory pool, and interpret that signal separately (UniProt O95967 localisation and processing; standard IF practice).
How do I reduce diffuse brown background without losing EFEMP2 deposits?
Inspect a no-primary control and a known positive section before changing antibody concentration, because diffuse chromogen can come from detection chemistry or tissue components (standard IHC practice). Block endogenous peroxidase for an enzyme-based chromogenic workflow, then verify that the DAB reaction is stopped consistently across sections (standard IHC practice). If background persists, adjust blocking, wash duration, and antibody dilution one variable at a time; the selected paraffin-tissue image reports 1:50 for this antibody (caption: A05653-1; standard IHC practice). Preserve finely distributed extracellular deposits during optimisation, since EFEMP2 is a matrix protein and HPA reports extracellular staining (UniProt O95967 localisation; HPA tissue IHC).
What should I score when EFEMP2 stains both cells and matrix? ⚠ ANSWER MARKED FOR VERIFICATION
Define cellular and extracellular regions before scoring, because HPA reports cytoplasmic expression plus extracellular deposits and EFEMP2 is secreted (HPA tissue IHC; UniProt O95967 localisation). For cells, report the percentage positive and, if intensity is reproducible, an H-score using 0–3 intensity grades (standard IHC scoring practice). For matrix, measure positive area or deposit density per mm² of evaluable tissue, with a fixed threshold and matched acquisition settings (standard IHC image-analysis practice). Normalise cellular counts to eligible cells and matrix measurements to eligible tissue area, excluding folds and necrosis by a prespecified rule (standard IHC scoring practice).
How can I distinguish convincing EFEMP2 signal from artefact?
Look for a reproducible extracellular distribution consistent with matrix or basement-membrane localisation, while allowing documented cytoplasmic staining (UniProt O95967 localisation; HPA tissue IHC). Treat isolated nuclear signal or staining restricted to section edges, folds, or necrotic areas as suspect until controls and serial sections support it (UniProt O95967 localisation; standard IHC practice). Compare the no-primary control to identify endogenous enzyme or detection-system signal, and check whether the pattern survives a consistent retrieval and DAB workflow (standard IHC practice). Do not infer that a stained cell produced adjacent matrix deposits: secreted protein can remain outside its source cell, and RNA–protein tissue correlation is complex (UniProt O95967 localisation; HPA tissue IHC).
Boster reagents
Best EFEMP2 / EGF-containing fibulin-like extracellular matrix protein 2 IHC Antibodies
A05653-1 has a paraffin-section IHC figure from human breast carcinoma (IHC image caption). IHC and IF are listed for human, mouse, and rat (catalog: A05653-1 applications/reactivity).
Real IHC data
Anti-Fibulin-4 (D123) EFEMP2 Antibody
Cat # A05653-1
A05653-1 is the only card that will render; its IHC figure shows paraffin-embedded human breast carcinoma at 1:50 (IHC image caption). It lists IHC and IF for human, mouse, and rat, but supplies no IF figure (catalog: A05653-1 applications/reactivity/IF image alts).
Which to pick: For tissue IHC, choose A05653-1 when its paraffin-section example is relevant; the caption does not report a fixative (IHC image caption). For IF, A05653-1 lists that application, but ICC validation is unreported (catalog: A05653-1 applications). For rat samples, A05653-1 lists rat reactivity while A05653-2 lists human and mouse; both are rabbit polyclonals, and A05653-2 has no supplied IHC figure (catalog: A05653-1 and A05653-2 reactivity/host/dilution_raw/IHC image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95967 (FBLN4_HUMAN, EGF-containing fibulin-like extracellular matrix protein 2).
- Human Protein Atlas. EFEMP2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. EFEMP2 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. EFEMP2 antibody validation summary (1 antibodies).
- EFEMP2 Is Associated with Shelterin-Related DNA Damage Repair, Immune Microenvironment Features, and Prognosis in Glioblastoma. Biomedicines 2026 — PMC13509514.
- EFEMP2 Inhibits Breast Cancer Invasion And Metastasis In Vitro And In Vivo. OncoTargets and therapy 2019 — PMC6826198.
- EFEMP2 suppresses epithelial-mesenchymal transition via Wnt/β-catenin signaling pathway in human bladder cancer. International journal of biological sciences 2019 — PMC6775297.
- EFEMP2 Suppresses the Invasion of Lung Cancer Cells by Inhibiting Epithelial-Mesenchymal Transition (EMT) and Down-Regulating MMPs. OncoTargets and therapy 2020 — PMC7034775.
- PubMed PMID:10601734 — UniProt-cited evidence.
- PubMed PMID:10982184 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EFEMP2 (EGF-containing fibulin-like extracellular matrix protein 2). Expected localisation Observed cytoplasm and extracellular deposits (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A05653-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Preleptotene spermatocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A05653-1); 2. Fixation: Image fixative and duration unreported (datasheet A05653-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 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-EFEMP2, 1:50-1:200 (datasheet A05653-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Extracellular deposits can complicate cell-level scoring (HPA tissue IHC); Isoform / epitope: No isoforms listed; no cytoplasmic domain; mature chain 24–443 (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt O95967, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A05653-1, PMC13509514, PMC6826198. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EFEMP2/immunohistochemistry-ihc-guide.