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AFF4 / AF4/FMR2 family member 4 · IHC design guide
Design Immunohistochemistry for AFF4
Plan AFF4 paraffin IHC around nuclear tissue staining (HPA tissue IHC) and a 2–5 μg/ml primary antibody range (datasheet A03824). Compare sections with consistent fixation and interpret staining cautiously because antibody staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AFF4 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 | Nuclear staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across many tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03824) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Ovary |
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 A03824) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Broad expression; low tissue specificity (HPA tissue IHC) | |
| Isoform / epitope | 3 isoforms; assess epitope coverage (UniProt) |
Section 1
Recommended AFF4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A03824) with the published human HNSCC tissue-array protocol (PMC6031063).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A03824) |
| Fixation | Image fixative and duration unreported (datasheet A03824); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03824); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03824) |
| Primary antibody | Rabbit anti-AFF4, 2-5μg/ml (datasheet A03824) |
| Primary incubation | Overnight at 4 °C (datasheet A03824) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03824) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AFF4-positive staining in glial cells of caudate (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A03824); the published protocol reports antigen retrieval without specifying its conditions (PMC6031063).
Section 2
What Is the Expected AFF4 Staining Pattern?
AFF4 should show predominantly nuclear staining in many cell types, including glial cells and round or early spermatids (HPA tissue IHC: ubiquitous nuclear expression; High in these cells). Its association with active chromatin and lack of a transmembrane segment support a nuclear interpretation (UniProt Q9UHB7: localization and topology). Treat tissue intensity as a guide, because HPA rates its IHC profile Approved while reporting low consistency between antibody staining and RNA expression.
| Nuclear chromogen in glial cells of caudate, cerebral cortex, or hippocampus, or in round or early spermatids. | This matches reported High staining in those cell populations (HPA tissue IHC). Assess signal within nuclei against the counterstain; compare intensity within the same run rather than treating High as a fixed score (general IHC practice). |
| Predominantly cytoplasmic or membrane staining, with nuclei largely clear. | This conflicts with the reported nuclear IHC profile (HPA tissue IHC) and nuclear/chromatin localization (UniProt Q9UHB7). Review controls and staining distribution before assigning such signal to AFF4; membrane-only staining is especially difficult to reconcile with its lack of a transmembrane segment (UniProt Q9UHB7 topology). |
| Strong staining in ovarian stroma cells while expected nuclear staining is absent elsewhere. | HPA reports AFF4 as Not detected in ovarian stroma cells (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity (general IHC practice); this observation does not establish that every cell in the ovary is negative. |
| Uniform chromogen over nuclei, cytoplasm, and surrounding tissue, with little cell-by-cell distinction. | A diffuse field does not reproduce the reported nuclear pattern (HPA tissue IHC). Evaluate background with detection controls and inspect whether the nuclear signal remains distinguishable after adjusting routine IHC conditions (general IHC practice). |
| No nuclear signal in a section containing an expected High-staining cell population. | For example, HPA reports High staining in testis round or early spermatids (HPA tissue IHC). Check that the relevant cells are present, then assess reagent performance and the staining run (general IHC practice); one blank section alone cannot establish absent AFF4 expression. |
💡Expected AFF4 appearanceCall a result consistent with AFF4 when chromogen is predominantly nuclear in the relevant cells, with conspicuous signal in HPA High populations such as glial cells or round or early spermatids (HPA tissue IHC); dominant cytoplasmic or membrane signal warrants investigation (UniProt Q9UHB7 localization and topology).
| How much confidence does the tissue IHC profile provide? | HPA describes ubiquitous nuclear expression and rates the IHC profile Approved, but notes low consistency between antibody staining and RNA expression (HPA tissue IHC). Use the profile to choose comparison cells, then interpret unexpected intensity cautiously. |
| How do cell populations affect comparison? | HPA reports High staining in selected glial cells and round or early spermatids, Medium in several other listed populations, and Not detected in ovarian stroma cells (HPA tissue IHC). Compare the named cell populations; a whole-organ label can hide different cellular results. |
| What should AFF4 look like by ICC-IF? | The main supported location is nucleoplasm, with additional supported localization in nucleoli fibrillar centers and nuclear bodies (HPA subcellular ICC-IF). That finer pattern comes from ICC-IF; it should not be required to score chromogenic tissue IHC. |
| Do isoforms define a distinct staining pattern? | UniProt lists three AFF4 isoforms (UniProt Q9UHB7). The supplied record gives no antibody epitope or isoform-specific IHC results, so isoform coverage and any isoform-specific tissue pattern cannot be determined here. |
| Does the protein record support surface or secreted staining? | AFF4 has no annotated signal peptide, propeptide, or transmembrane segment (UniProt Q9UHB7 processing and topology). These annotations support scrutiny of dominant surface staining; they do not establish an antigen retrieval setting or a fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected High-staining cells have no nuclear signal. | The relevant cells may be missing from the viewed area, or the staining run may have failed (HPA tissue IHC: High in specified cells; general IHC practice). | Confirm cell identity on the section; inspect a known-positive comparison processed in the same run, then review the antibody and detection steps (general IHC practice). |
| Signal is strongest at membranes or throughout cytoplasm. | The pattern conflicts with AFF4 nuclear localization and lack of a transmembrane segment (UniProt Q9UHB7); nonspecific binding is one possibility (general IHC practice). | Check whether nuclei stain in an expected-positive population and compare with detection controls before interpreting the extranuclear signal (HPA tissue IHC; general IHC practice). |
| Ovarian stroma cells stain strongly. | HPA reports these cells as Not detected; cross-reactivity or endogenous detection activity are possible explanations (HPA tissue IHC; general IHC practice). | Inspect a control omitting primary antibody and review cell identity and signal localization; do not label the whole ovary negative from the stromal observation (general IHC practice; HPA tissue IHC). |
| The entire section has diffuse chromogenic background. | Nuclear AFF4 staining may be obscured by nonspecific binding or background from the detection system (HPA tissue IHC: nuclear profile; general IHC practice). | Compare primary-omission and positive controls; review blocking, antibody concentration, washing, and chromogen development as routine IHC variables (general IHC practice). |
| A tissue seems weaker than its RNA expression would suggest. | HPA explicitly reports low consistency between antibody staining and RNA expression despite its Approved IHC profile (HPA tissue IHC). | Score the observed cell population and nuclear pattern directly; use an expected-positive comparison in the same run before calling the tissue negative (HPA tissue IHC; general IHC practice). |
| Fine nucleolar or nuclear-body detail is unclear on a chromogenic section. | Those additional locations are supported by ICC-IF images, whereas tissue IHC is summarized as nuclear (HPA subcellular ICC-IF; HPA tissue IHC). | Score the nuclear IHC result without requiring resolution of those subnuclear structures; consult the separate ICC-IF guide for fluorescence interpretation (HPA tissue IHC; HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing AFF4 staining
A03824 · IHCIHC analysis of AFF4 using anti-AFF4 antibody (A03824). AFF4 was detected in a paraffin-embedded section of human placenta tissue. Heat medi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000072364-AFF4/tissue/cerebral+cortex#imid_6082184
HPA · HippocampusHuman Protein Atlas IHC, Hippocampus. Source: https://www.proteinatlas.org/ENSG00000072364-AFF4/tissue/hippocampal+formation#imid_6082185
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000072364-AFF4/tissue/colon#imid_6082118
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000072364-AFF4/tissue/liver#imid_6082103
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA029634; green = AFF4, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA029634; green = AFF4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AFF4 IHC & IF
🧪Run caudate first and score glial cell nuclei for AFF4 staining (HPA: High in caudate glial cells; UniProt Q9UHB7: nucleus). Use ovarian stroma as the negative tissue (HPA: Not detected in ovarian stroma cells); on the caudate slide, cells without nuclear signal can serve as background comparators, but the supplied HPA row does not identify a specific internal negative cell type.
Positive control tissue: Caudate (Glial cells, HPA High)
Negative control tissue: Ovary (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AFF4 in CACO-2, SiHa, U2OS, U-251MG, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody), and AFF4 knockout material as a biological specificity control. Quench endogenous peroxidase and inspect caudate sections for pigment that could be mistaken for chromogenic signal (standard IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section caption reports heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required across specimens; its fixative is unreported (selected-SKU tissue-IHC caption). A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the HPA staining rows do not establish specimen processing. HPA reports nucleoplasmic ICC-IF localization (HPA: supported nucleoplasm), but the supplied evidence does not establish whether IF or frozen sections are easier than paraffin IHC; inspect caudate tissue for pigment during chromogenic scoring (standard IHC practice).
HPA tissue IHC evidence for AFF4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | High | Protein (IHC) | HPA → |
| Testis | Round or early spermatids | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AFF4 IHC Tips
Troubleshoot AFF4 staining in paraffin sections by checking retrieval, nuclear localisation and cell-level controls before interpreting chromogenic signal.
Where should I start if AFF4 nuclear staining is weak after retrieval?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03824). The selected paraffin-section example used this retrieval before staining human placenta with 2 μg/ml primary antibody overnight at 4°C (datasheet A03824). Compare a retrieved section with a matched section processed without retrieval, keeping detection and counterstaining identical (standard IHC practice). If nuclei remain weak, adjust heating and cooling within the tissue’s tolerance, then consider another retrieval buffer as a fallback; record any increase in diffuse background (standard IHC practice). Score nuclear signal separately from cytoplasmic colour, since AFF4 is mainly nucleoplasmic (HPA subcellular: nucleoplasm supported).
How can I troubleshoot weak AFF4 staining when fixation conditions vary?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet A03824). Record the actual fixative, fixation duration, tissue thickness and processing history for each specimen before comparing nuclear staining (standard IHC practice). On matched material, hold EDTA retrieval at pH 8.0 and the primary incubation at 2 μg/ml overnight at 4°C while assessing fixation-related variation (datasheet A03824). Inspect morphology and the distribution of nuclear signal together, because damaged or poorly preserved regions can confound staining comparisons (standard IHC practice). Do not assign a fixation effect to AFF4 without a controlled comparison.
Should AFF4 staining be nuclear, and how should I assess cytoplasmic colour?
Expect predominantly nuclear staining: AFF4 associates with transcriptionally active chromatin (UniProt Q9UHB7: localisation), and supported subcellular imaging places it mainly in the nucleoplasm, with additional nuclear-body and nucleolar fibrillar-center localisation (HPA subcellular: supported locations). Use the counterstain to check whether chromogen lies inside intact nuclei, and inspect cytoplasmic colour separately (standard IHC practice). AFF4 has no annotated transmembrane segment or signal peptide, so a dominant membrane rim lacks support from its recorded topology (UniProt Q9UHB7: topology and processing). Compare the suspected pattern with a no-primary control and with other cells on the same section (standard IHC practice). Report mixed patterns by compartment rather than calling all colour AFF4-positive.
Could an AFF4 isoform or modified epitope explain discordant staining?
AFF4 has 3 annotated isoforms and multiple modified residues, including phosphoserines at positions 120, 212, 387, 388 and 389 (UniProt Q9UHB7: isoforms and modified residues). Those annotations do not identify the catalog antibody’s epitope or establish which isoforms it detects. Request its immunogen or epitope information, then map that sequence against each isoform before attributing a staining difference to splice variation (standard antibody-validation practice). Compare suspect sections under identical EDTA pH 8.0 retrieval and primary-antibody conditions (datasheet A03824). If localisation or intensity still differs, seek an independently validated antibody recognising another region and retain the original result as epitope-dependent until confirmed (standard IHC practice).
How should I adapt AFF4 assessment to multiplex immunofluorescence?
Treat IF as a separate validation: the selected catalog-antibody example documents chromogenic staining of a paraffin section, without IF conditions (datasheet A03824). Co-stain with a validated marker for the cell population being assessed and a nuclear counterstain, then score AFF4 within those marked nuclei (standard IF practice; HPA tissue IHC: cell-dependent staining). Choose a fluorophore channel after measuring tissue autofluorescence and include single-colour controls to check bleed-through (standard IF practice). Because AFF4 is mainly nucleoplasmic and has no transmembrane segment, use permeabilisation suitable for a nuclear epitope, optimised against morphology and background (HPA subcellular: nucleoplasm supported; UniProt Q9UHB7: topology; standard IF practice). Do not transfer the paraffin-section retrieval settings to IF without testing them.
What should I check when DAB colour obscures AFF4-positive nuclei?
The selected paraffin-section example used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (datasheet A03824). Run no-primary and secondary-only controls to identify detection-system staining; include a peroxidase block as a general chromogenic IHC step (standard IHC practice). Shorten DAB development or reduce primary exposure if colour spreads beyond nuclear boundaries, checking that nuclear signal remains visible (standard IHC practice). Inspect tissue edges, folds and damaged areas separately, since accumulated reagent can mimic a focal positive pattern (standard IHC practice). Judge diffuse cytoplasmic colour cautiously against AFF4’s mainly nucleoplasmic localisation (HPA subcellular: nucleoplasm supported).
How should I score heterogeneous AFF4 staining across a tissue section? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear scoring threshold before reviewing experimental groups, using the same counterstain and imaging settings throughout (standard IHC practice). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score from intensity categories 0–3, with a possible range of 0–300 (standard IHC scoring practice). Normalise counts to the number of evaluable nuclei in the defined compartment, and report sampled area or density per mm² if cell abundance itself matters (standard IHC practice). AFF4 expression varies by cell population in tissue IHC, so do not pool unlike compartments into one denominator (HPA tissue IHC: cell-level results). Exclude folds, edges and necrotic regions by a prespecified rule (standard IHC practice).
How can I distinguish genuine AFF4 staining from artefact?
A credible result places chromogen in intact nuclei, consistent with AFF4’s nuclear and chromatin-associated localisation (UniProt Q9UHB7: localisation; HPA subcellular: nucleoplasm supported). Compare staining with expected cell populations, while allowing that HPA reports variable cell-level intensity and absent detection in ovarian stroma cells (HPA tissue IHC: cell-level results). Treat edge-only colour, necrotic areas, membrane rims and signal persisting in a no-primary control as reasons to investigate artefact; check peroxidase blocking when background persists (standard IHC practice; UniProt Q9UHB7: topology). Recheck discordant cases with matched processing and an independent antibody where available (standard IHC practice). Interpret agreement with expression data cautiously because the HPA tissue-IHC assessment reports low staining–RNA consistency (HPA tissue IHC: Approved reliability description).
Boster reagents
Best AFF4 / AF4/FMR2 family member 4 IHC Antibodies
The catalog includes one anti-AFF4 antibody with real IHC data from a human placenta paraffin section (A03824 image caption); its listed reactivity covers human, mouse and rat (A03824 catalog).
Real IHC data
Anti-AFF4 Antibody ®
Cat # A03824
A03824 will render with an IHC figure from a human placenta paraffin section (A03824 image caption). IHC is a listed application, and human, mouse and rat are listed as reactive species (A03824 catalog).
Which to pick: Choose A03824 for paraffin-section IHC: its own caption documents EDTA retrieval at pH 8.0 and staining at 2 μg/ml in human placenta (A03824 image caption); the fixative is unreported (A03824 image caption). A03824 lists human, mouse and rat reactivity, but its supplied IHC figure documents human tissue only (A03824 catalog; A03824 image caption). No IF/ICC application or IF figure is supplied for A03824, so there is no catalog-supported IF/ICC pick here (A03824 catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UHB7 (AFF4_HUMAN, AF4/FMR2 family member 4).
- Human Protein Atlas. AFF4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. AFF4 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nucleoli fibrillar center and nuclear bodies..
- Human Protein Atlas. AFF4 antibody validation summary (3 antibodies).
- AFF4 Predicts the Prognosis of Colorectal Cancer Patients and Suppresses Colorectal Cancer Metastasis via Promoting CDH1 Expression. Frontiers in oncology 2022 — PMC8865618.
- Impact of disulfidptosis-associated clusters on breast cancer survival rates and guiding personalized treatment. Frontiers in endocrinology 2023 — PMC10728640.
- AFF4 promotes tumorigenesis and tumor-initiation capacity of head and neck squamous cell carcinoma cells by regulating SOX2. Carcinogenesis 2018 — PMC6031063.
- AFF4 promotes tumor progression and cisplatin resistance by modulating the PTEN/PI3K/AKT/mTOR axis to accelerate glycolysis in lung adenocarcinoma. Cell & bioscience 2025 — PMC12337439.
- PubMed PMID:10588740 — UniProt-cited evidence.
- PubMed PMID:12065898 — UniProt-cited evidence.
- PubMed PMID:15372022 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AFF4 (AF4/FMR2 family member 4). Expected localisation Nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03824). Validated IHC antibody: A03824 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Glial cells); negative Ovary. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A03824); 2. Fixation: Image fixative and duration unreported (datasheet A03824); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A03824); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03824); 8. Primary antibody: Rabbit anti-AFF4, 2-5μg/ml (datasheet A03824); 9. Primary incubation: Overnight at 4 °C (datasheet A03824). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03824); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; assess epitope coverage (UniProt); Regulation: Broad expression; low tissue specificity (HPA tissue IHC). Evidence: UniProt Q9UHB7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A03824, PMC6031063. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AFF4/immunohistochemistry-ihc-guide.