This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RELA / Transcription factor p65 · IHC design guide
Design Immunohistochemistry for RELA
Plan chromogenic RELA IHC on paraffin sections using PA1669 at 2–5 μg/mL (datasheet: PA1669). Assess the widespread cytoplasmic tissue pattern (HPA tissue IHC) while allowing for nuclear translocation after activation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RELA Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PA1669) | |
| Positive control | Appendix+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 | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Activation can shift RELA into nuclei (UniProt) | |
| Regulation | Activation shifts nuclear staining (UniProt) | |
| Isoform / epitope | Four isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended RELA IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet PA1669). Published RELA IHC protocols cover ovine thymus (PMC9587240) and mouse renal tissue (PMC9892181).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet PA1669) |
| Fixation | Image fixative and duration unreported (datasheet PA1669); 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 PA1669); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PA1669) |
| Primary antibody | Rabbit anti-RELA, 2-5μg/ml (datasheet PA1669) |
| Primary incubation | Overnight at 4 °C (datasheet PA1669) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PA1669) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RELA-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet PA1669). Citrate pH 6.0 is a published alternative for ovine thymus (PMC9587240).
Section 2
What Is the Expected RELA Staining Pattern?
RELA is mainly cytoplasmic in the tissue IHC profile, with nuclear localisation possible after activation (HPA: ubiquitous cytoplasmic expression; UniProt Q04206: nucleus and cytoplasm). Expect staining across many cell types, including strong staining in appendix lymphoid tissue, bone marrow hematopoietic cells, and several glandular epithelia (HPA: High in these cells). The tissue profile is Supported, with medium consistency between staining and RNA data (HPA: reliability Supported). RELA has no transmembrane segment (UniProt Q04206 topology).
| Cytoplasmic staining across many cells, with strong signal in appendix lymphoid tissue or bone marrow hematopoietic cells. | This fits the reported tissue pattern; compare intensity within the same run because staining can vary by cell type (HPA: ubiquitous cytoplasmic expression; High in these cells; standard IHC practice). |
| Nuclear staining appears in a subset of otherwise plausible positive cells. | Nuclear RELA can reflect activation and translocation; nuclear staining alone does not establish pathway activation without a suitable experimental comparison (UniProt Q04206: cytoplasmic inhibitor complex and nuclear translocation). |
| Signal is confined to cell membranes or extracellular material, with no convincing intracellular staining. | That distribution conflicts with the reported cytoplasmic and nuclear locations and warrants an artefact check; RELA has no transmembrane segment (HPA: cytoplasmic expression; UniProt Q04206 topology and subcellular location). |
| Strong staining appears in a cell population expected to be weak, while nearby expected-positive cells are unstained. | Consider cross-reactivity or endogenous chromogenic detection activity before assigning the signal to RELA; some populations have low reported staining, but HPA does not define them as negative controls (HPA: Low in selected cells; standard IHC practice). |
| Colour covers much of the section without clear cell boundaries, or a known high-staining tissue has no signal. | Diffuse colour suggests background, while absent signal calls for a control and detection check; neither observation alone establishes RELA absence (HPA: High in appendix lymphoid tissue and bone marrow hematopoietic cells; standard IHC practice). |
💡Expected RELA appearanceCall a result positive when cytoplasmic RELA staining is cell associated, with strong signal in an HPA high-staining population and possible nuclear signal in an appropriate activation context; isolated membrane or diffuse extracellular colour is suspect (HPA: tissue profile; UniProt Q04206: nucleus, cytoplasm, no transmembrane segment).
| Activation state and compartment | Inactive RELA can be retained in the cytoplasm by I-kappa-B; activation can release the complex for nuclear translocation, so record cytoplasmic and nuclear staining separately (UniProt Q04206 function and subcellular location). |
| Tissue and cell selection | Appendix lymphoid tissue, bone marrow hematopoietic cells, and breast or colon glandular cells are reported High; selected endothelial, glial, respiratory, and other cells are Low (HPA: tissue IHC). |
| Antibody evidence | CAB004264 and CAB005030 each have Supported IHC status; HPA calls the overall tissue profile Supported with medium staining–RNA consistency, so retain tissue controls when interpreting an unexpected pattern (HPA: antibody validation; tissue reliability). |
| Isoforms and epitope | Four RELA isoforms are listed, but this payload gives no antibody epitope or isoform coverage; an isoform-specific explanation for a staining difference is therefore unestablished (UniProt Q04206: four isoforms). |
| IF/ICC Q: Where should RELA appear? | A: Cytosolic signal is the reported baseline, with nuclear localisation biologically possible after stimulation; assess IF/ICC on its own guide page (HPA: Cytosol, enhanced, in ICC-IF; UniProt Q04206: nuclear translocation). |
| Situation | Likely cause | Next action |
|---|---|---|
| Only membrane-like or extracellular colour is visible. | The distribution does not match reported RELA compartments; nonspecific deposition or detection artefact is possible (UniProt Q04206 topology and location; standard IHC practice). | Check the no-primary control and inspect whether colour follows cell interiors before scoring RELA (standard IHC practice). |
| Nuclear staining seems unexpectedly strong. | RELA can enter nuclei after activation, so the compartment alone cannot distinguish activation from an assay problem (UniProt Q04206 function). | Score nuclear and cytoplasmic compartments separately and compare matched conditions plus controls; avoid calling activation from one section alone (standard IHC practice; UniProt Q04206 function). |
| A nominally low-staining cell type shows strong colour. | Cell-type assignment may be wrong, or antibody cross-reactivity or endogenous detection activity may contribute (HPA: Low in selected cell types; standard IHC practice). | Recheck morphology and the HPA cell-level entry; examine no-primary and detection controls before attributing the colour to RELA (HPA: tissue IHC; standard IHC practice). |
| The whole section has diffuse chromogenic colour. | Background may arise from insufficient blocking, incomplete washing, or endogenous detection activity (standard IHC practice). | Inspect negative controls, then optimise blocking, washes, antibody dilution, and detection conditions using the assay's instructions (standard IHC practice). |
| Appendix lymphoid tissue or bone marrow hematopoietic cells show no convincing signal. | These are reported High, making an assay or specimen issue plausible; an absent result alone cannot identify its cause (HPA: High in these cells; standard IHC practice). | Verify positive-control staining and run records; review retrieval, antibody dilution, detection reagents, and counterstain without assuming a RELA-specific fixation effect (standard IHC practice). |
| Two sections give different proportions of nuclear and cytoplasmic staining. | Activation context can alter RELA location, while section and detection variation can complicate comparison (UniProt Q04206 function; standard IHC practice). | Compare equivalent cell populations under matched staining conditions, document each compartment, and interpret differences against experimental controls (standard IHC practice). |
Experiment evidence
IHC & IF images showing RELA staining
PA1669 · IHCIHC analysis of NF-kB p65/RELA using anti-NF-kB p65/RELA antibody (PA1669). NF-kB p65/RELA was detected in a paraffin-embedded section of hu
PA1669 · IFIF analysis of NF-kB p65/RELA using anti-NF-kB p65/RELA antibody (PA1669). NF-kB p65/RELA was detected in an immunocytochemical section of U
M00284-4 · IHCMouse colon was stained with anti-NF-κB p65 rabbit antibody
M00284-4 · IHCHuman colon was stained with anti-NF-κB p65 rabbit antibody
M00284 · IHCImmunohistochemical analysis of paraffin-embedded human transitional cell carcinoma of bladder, using NF-κB p65 Antibody.
M00284 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
A00284-1 · IFIF analysis of NF-kB p65 using anti-NF-kB p65 antibody (A00284-1). NF-kB p65 was detected in an immunocytochemical section of U2OS cells. En
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000173039-RELA/tissue/cerebral+cortex#imid_2115560
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000173039-RELA/tissue/colon#imid_2083510
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000173039-RELA/tissue/liver#imid_2083519
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000173039-RELA/tissue/kidney#imid_2083520
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004264; green = RELA, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004264; green = RELA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RELA IHC & IF
🧪Run breast first and expect glandular cells to stain (HPA: High in breast glandular cells). HPA detects RELA in all 45 scored tissues, so there is no validated negative tissue; no-primary and isotype controls carry the negative comparison, while any unstained cells on the positive slide should show only background chromogen and should not be designated RELA-negative (HPA: detected in all 45 scored tissues).
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: None in HPA: RELA 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 RELA in A-431, U-251MG, U2OS, A-549, PC-3, with annotated localisation: Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a concentration-matched rabbit IgG isotype control for the rabbit catalog antibody (caption: rabbit primary and anti-rabbit secondary); use matched RELA-knockout material as a biological negative if available (standard IHC specificity control). Block endogenous peroxidase and check background in the breast section before interpreting DAB staining (caption: breast section with peroxidase/DAB detection; standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PA1669 paraffin-section caption does not state a fixative (caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; the supplied evidence also does not establish whether frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images). No breast-specific artefact is reported in the supplied evidence; assess background in glandular and surrounding tissue when scoring (HPA: High in breast glandular cells; standard IHC practice).
HPA tissue IHC evidence for RELA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: RELA is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RELA IHC Tips
Use compartment-aware controls when optimizing chromogenic RELA IHC; nuclear staining and cytoplasmic staining answer different biological questions (UniProt Q04206; HPA tissue IHC).
How should I optimize retrieval when RELA staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PA1669). The selected tissue image used this condition before staining with 2 μg/ml antibody overnight at 4°C, so keep those variables fixed while assessing retrieval (datasheet PA1669). Compare adjacent sections processed with the same heating and cooling schedule, and include a section without primary antibody to identify detection background (standard IHC practice). If staining remains weak, adjust retrieval duration incrementally and inspect tissue preservation alongside staining intensity (standard IHC practice). Score cytoplasmic and nuclear signal separately because inactive RELA can remain cytoplasmic whereas activated complexes can enter nuclei (UniProt Q04206).
Could fixation explain weak or inconsistent RELA staining?
The selected paraffin-section caption does not report a fixative, and target-specific RELA sensitivity to fixation is unknown here (datasheet PA1669). Record the fixative, fixation duration, section age and processing history for each specimen before comparing staining results (standard IHC practice). Process a reference section alongside experimental sections, then hold retrieval at EDTA pH 8.0 and antibody concentration at 2 μg/ml while investigating variability (datasheet PA1669; standard IHC practice). Examine morphology as well as signal, since damaged or poorly preserved regions can distort compartment scoring (standard IHC practice). Do not infer fixation tolerance from the protein’s cytoplasmic and nuclear distribution or from tissue staining patterns (UniProt Q04206; HPA tissue IHC).
Why do some cells stain in the cytoplasm while others show nuclear RELA?
Cytoplasmic RELA is biologically plausible because inactive NF-κB complexes can be retained there by I-κB (UniProt Q04206). Nuclear RELA is also plausible after pathway activation, when liberated complexes translocate to the nucleus (UniProt Q04206). The tissue atlas describes ubiquitous cytoplasmic staining, while its cell imaging summary identifies cytosolic localization (HPA tissue IHC; HPA subcellular). For chromogenic IHC, score nuclear and cytoplasmic staining separately within the same annotated cell population and inspect the counterstain to confirm nuclear boundaries (standard IHC practice). Compare similarly processed sections and include a negative detection control before interpreting a shift in nuclear fraction as pathway activity (standard IHC practice).
How can epitope choice affect RELA staining across specimens?
RELA has 4 annotated isoforms and an RHD spanning residues 19–306, so record the antibody’s stated immunogen or epitope before comparing specimens (UniProt Q04206; standard IHC practice). The supplied product caption establishes paraffin-section staining but does not locate the recognized epitope or establish isoform coverage (datasheet PA1669). RELA also has annotated modified residues, making epitope accessibility a consideration when a site overlaps the antibody-binding region (UniProt Q04206; standard IHC practice). Compare staining with a second antibody against a documented distinct region, if available, while keeping section processing and scoring consistent (standard IHC practice). Avoid assigning an isoform or modification state from total chromogenic staining alone (standard IHC practice).
How should I follow up a RELA IHC localization result by IF?
Use IF/ICC as a separate localization follow-up to the paraffin-section chromogenic result, which used DAB detection (datasheet PA1669; standard IHC practice). Multiplex RELA with a marker identifying the expected cell population, such as the glandular cells being evaluated in breast tissue, and assess nuclear overlap with a DNA counterstain (HPA tissue IHC; standard IF practice). Select spectrally separated fluorophores and inspect an unstained specimen for tissue autofluorescence before choosing channels (standard IF practice). RELA has no annotated transmembrane segment and occurs inside cells, so permeabilize as appropriate for access to its cytoplasmic or nuclear epitope (UniProt Q04206; standard IF practice). Optimize IF antibody conditions independently of the 2 μg/ml chromogenic IHC condition (datasheet PA1669; standard IF practice).
What should I check when RELA DAB staining looks diffuse?
First compare a section without primary antibody to the stained section to locate background from the detection system (standard IHC practice). The selected workflow used a peroxidase-conjugated secondary antibody and DAB; blocking endogenous peroxidase is a general chromogenic IHC step (datasheet PA1669; standard IHC practice). Check whether the 10% goat serum block, 2 μg/ml primary concentration and overnight incubation at 4°C were applied consistently (datasheet PA1669). Review wash stringency and DAB development time while preserving a common reference section across runs (standard IHC practice). Diffuse cytoplasmic signal alone is not automatically background, because inactive RELA can reside in the cytoplasm (UniProt Q04206).
How should I quantify nuclear RELA in heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and representative viable regions before scoring, then apply the same inclusion rules to every section (standard IHC practice). Record the percentage of cells with nuclear DAB staining and, separately, an intensity-weighted nuclear H-score; retain cytoplasmic scores as a distinct readout (standard IHC practice). Normalize positive-cell counts to the number of eligible cells, or report density per mm² of viable tissue when cell counting is impractical (standard IHC practice). Keep retrieval at EDTA pH 8.0 and detection settings consistent across compared sections (datasheet PA1669; standard IHC practice). Because RELA can occupy either compartment, a total staining score can obscure a biologically relevant change in nuclear fraction (UniProt Q04206).
How can I distinguish genuine RELA signal from staining artefacts?
Interpret RELA staining in the annotated cell type and compartment: cytoplasmic retention and nuclear translocation are both biologically plausible (UniProt Q04206). Check the counterstain for intact nuclei and exclude necrotic areas or section edges where staining and morphology are unreliable (standard IHC practice). Compare a section without primary antibody and review endogenous peroxidase blocking before attributing DAB deposits to RELA (standard IHC practice). The atlas reports high staining in breast glandular cells, among other listed populations, but its overall tissue-IHC reliability is Supported with medium RNA agreement (HPA tissue IHC). Treat nuclear staining as evidence of localization, and require an appropriate functional readout before claiming NF-κB transcriptional activation (UniProt Q04206; standard IHC practice).
Boster reagents
Best RELA / Transcription factor p65 IHC Antibodies
The catalog includes IHC images from human breast cancer, human bladder carcinoma, and mouse colon, plus an IF image from U2OS cells (catalog figure captions). Listed antibody reactivity spans human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-NF-kB p65/RELA Antibody ®
Cat # PA1669
Real IHC data
Anti-NF-κB p65 Rabbit Monoclonal Antibody
Cat # M00284-4
Real IHC data
Anti-NF-Kappa B p65 RELA Rabbit Monoclonal Antibody
Cat # M00284
Real IF data
Anti-NF-kB p65/RELA Antibody ®
Cat # A00284-1
PA1669 has paraffin-section IHC data from human breast cancer, while M00284 has paraffin-section IHC data from human transitional cell carcinoma of the bladder (respective IHC figure captions). M00284-4 has mouse-colon IHC data, and A00284-1 has U2OS-cell IF data (respective figure captions).
Which to pick: For paraffin-section tissue IHC, choose PA1669 for the documented human breast cancer workflow or M00284 for the documented human bladder example; the fixative is unreported in both captions (respective IHC figure captions). For IF/ICC, choose A00284-1, which lists both applications and shows staining in U2OS cells (catalog applications; A00284-1 IF figure caption). For cross-species IHC planning, consider M00284-4: it is a monoclonal antibody listed as reactive with human, mouse, and rat, with IHC images from mouse colon, human colon, and human tonsil; those captions do not report the fixative (M00284-4 catalog description, reactivity, and IHC figure captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q04206 (TF65_HUMAN, Transcription factor p65).
- Human Protein Atlas. RELA tissue IHC expression (reliability: Supported).
- Human Protein Atlas. RELA subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. RELA antibody validation summary (3 antibodies).
- Changes in expression of nuclear factor kappa B subunits in the ovine thymus during early pregnancy. Scientific reports 2022 — PMC9587240.
- Supratentorial ependymoma in childhood: more than just RELA or YAP. Acta neuropathologica 2021 — PMC7882569.
- LDP alleviates TKI-induced proteinuria through reversing the expression of RelA in renal tissues. Frontiers in medicine 2023 — PMC9892181.
- Diagnostics of pediatric supratentorial RELA ependymomas: integration of information from histopathology, genetics, DNA methylation and imaging. Brain pathology (Zurich, Switzerland) 2019 — PMC7379587.
- PubMed PMID:2006423 — UniProt-cited evidence.
- PubMed PMID:8281153 — UniProt-cited evidence.
- PubMed PMID:7907305 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RELA (Transcription factor p65). Expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PA1669). Validated IHC antibody: PA1669 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet PA1669); 2. Fixation: Image fixative and duration unreported (datasheet PA1669); 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 PA1669); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PA1669); 8. Primary antibody: Rabbit anti-RELA, 2-5μg/ml (datasheet PA1669); 9. Primary incubation: Overnight at 4 °C (datasheet PA1669). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Activation can shift RELA into nuclei (UniProt); Isoform / epitope: Four isoforms; verify epitope coverage (UniProt); Regulation: Activation shifts nuclear staining (UniProt). Evidence: UniProt Q04206, HPA tissue IHC · Supported, HPA ICC-IF · cytosol., Datasheet PA1669, PMC9587240, PMC9892181. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RELA/immunohistochemistry-ihc-guide.