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RFX1 / MHC class II regulatory factor RFX1 · IHC design guide
Design Immunohistochemistry for RFX1
Plan chromogenic RFX1 IHC around nuclear staining and a 2–5 μg/mL antibody starting range (HPA tissue IHC; datasheet A04392-1). Use colon glandular cells as a positive reference and adipocytes as an undetected comparator (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RFX1 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 most tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear signal in most tissues, including glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04392-1) | |
| Positive control | Colon+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 conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | Regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms or alternate processing annotated (UniProt) |
Section 1
Recommended RFX1 IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by published RFX1 protocols for esophageal, liver, and colorectal specimens (PMC3174211; PMC11996997; PMC12712669).
| Sample | Paraffin-embedded Diffuse large B-cell lymphoma of human intestine tissue; fixative not specified (datasheet A04392-1) |
| Fixation | Image fixative and duration unreported (datasheet A04392-1); 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 A04392-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04392-1) |
| Primary antibody | Rabbit anti-RFX1, 2-5 μg/ml (datasheet A04392-1) |
| Primary incubation | Overnight at 4 °C (datasheet A04392-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04392-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RFX1-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A04392-1). Other antibodies were used without retrieval or with citrate at pH 6.0 (PMC3174211; PMC12712669).
Section 2
What Is the Expected RFX1 Staining Pattern?
RFX1 is a nuclear, DNA-binding protein with no transmembrane segment (UniProt P22670: subcellular location, function, topology). In paraffin-section IHC, expect nuclear staining in many tissues, especially colon, rectum, and stomach glandular cells and kidney tubular cells (HPA: nuclear expression in most tissues; High in these cells). HPA rates its tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression (HPA: reliability description).
| Distinct nuclear staining in colon, rectum, or stomach glandular cells, or kidney tubular cells. | This matches the reported High cell-specific staining and nuclear tissue profile (HPA: tissue IHC). Assess the named cells against nearby nuclei and background; a positive tissue section establishes that the run can detect the expected pattern, but does not by itself establish antibody specificity (HPA: IHC Approved). |
| Predominantly cytoplasmic, membranous, or broadly extracellular chromogen. | A dominant signal outside nuclei conflicts with RFX1's nuclear location and lack of a transmembrane segment (UniProt P22670: subcellular location, topology). Treat it as suspect until nuclear staining appears in an HPA High cell population and the pattern is checked against a no-primary control (HPA: tissue IHC; general IHC practice). |
| Strong staining in adipocytes, prostate glandular cells, or smooth muscle cells. | These specific cell populations are reported as Not detected in HPA tissue IHC. Unexpected staining raises possible cross-reactivity or endogenous detection activity; compare the cell identity, distribution, and no-primary control before assigning it to RFX1 (HPA: tissue IHC; general IHC practice). |
| Weak haze across nuclei and surrounding tissue, without a clear cell-specific pattern. | Diffuse background cannot be scored as the reported nuclear expression in most tissues (HPA: tissue IHC). Review background in the no-primary control and reassess blocking, washing, antibody concentration, and detection exposure as general chromogenic IHC checks; none establishes an RFX1-specific cause (general IHC practice). |
| No nuclear signal in colon glandular cells or kidney tubular cells. | This misses two HPA High reference populations and makes a negative test section difficult to interpret (HPA: tissue IHC). First confirm the expected cells are present, then check the IHC-P run and controls; absence alone cannot distinguish a technical failure from biological variation (general IHC practice). |
💡Expected RFX1 appearanceCall an IHC section positive when colon, rectum, or stomach glandular cells or kidney tubular cells show clear, predominantly nuclear staining consistent with HPA High staining; dominant staining outside nuclei or strong staining in HPA Not detected cell populations is suspect (HPA: tissue IHC; UniProt P22670: nucleus).
| Reference tissue and cell identity | Use the named cell populations, rather than an entire organ, to judge the pattern: HPA reports High staining in colon, rectum, and stomach glandular cells and kidney tubular cells, but Not detected in several other cell populations (HPA: tissue IHC). |
| Evidence strength | HPA describes nuclear expression in most tissues and rates its IHC profile Approved, while flagging low consistency between antibody staining and RNA expression; interpret a matching image as supporting evidence, with that reliability caveat (HPA: tissue IHC reliability). |
| Cellular location and topology | UniProt places RFX1 in the nucleus and annotates no transmembrane segment or signal peptide. These features support a nuclear interpretation, but do not identify an antibody epitope or predict how paraffin-section processing affects detection (UniProt P22670: subcellular location, topology, processing). |
| Antigen retrieval | Retrieval conditions may be compared during paraffin IHC optimization (general IHC practice). No RFX1-specific retrieval requirement or fixation sensitivity is supplied here, so a change in staining after retrieval must be judged using the slide controls rather than attributed to a documented RFX1 effect. |
| IF/ICC Q&A: where should signal appear? | Mainly in the nucleoplasm (HPA: ICC-IF, enhanced); HPA also lists vesicles as an uncertain additional location. This IF/ICC observation is a localization cross-check, not a paraffin IHC expectation or an IF protocol (HPA: subcellular summary). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular or tubular cells show no nuclear stain. | The result conflicts with HPA High staining in colon, rectum, and stomach glandular cells or kidney tubular cells; the cause is unresolved (HPA: tissue IHC). | Verify that the expected cells and a working detection control are present; review the IHC-P run's retrieval, antibody application, and detection steps as general IHC checks. Repeat before interpreting a test tissue as negative (general IHC practice). |
| Signal is strongest in cytoplasm or along cell borders. | That distribution conflicts with the reported nuclear location and no-transmembrane topology (UniProt P22670: subcellular location, topology). | Compare nuclear and extranuclear staining in an HPA High reference population and inspect a no-primary control. If the extranuclear pattern persists, withhold a positive RFX1 call pending antibody-specific validation (HPA: tissue IHC; general IHC practice). |
| Adipocytes, prostate glandular cells, or smooth muscle cells stain strongly. | HPA reports these cell populations as Not detected; cross-reactivity or detection background is possible, but the image alone cannot identify which (HPA: tissue IHC). | Confirm cell identity, compare with a nuclear positive reference, and run a no-primary control to assess detection background (HPA: tissue IHC; general IHC practice). |
| The whole section has diffuse brown background. | General IHC possibilities include nonspecific antibody binding, incomplete washing, or endogenous detection activity; no RFX1-specific background mechanism is supplied (general IHC practice). | Inspect a no-primary control, then review blocking, washes, detection conditions, and antibody concentration. Score only a resolved nuclear, cell-specific pattern (general IHC practice; UniProt P22670: nucleus). |
| Staining varies between tissue types or disagrees with RNA data. | HPA explicitly reports low consistency between antibody staining and RNA expression, despite its Approved tissue IHC rating (HPA: reliability description). | Record the exact tissue and cell population, compare with HPA's cell-level staining categories, and retain the discrepancy in the interpretation rather than treating RNA and staining as interchangeable measures (HPA: tissue IHC reliability). |
| Vesicular IF/ICC signal is used to justify cytoplasmic IHC staining. | HPA calls vesicles an uncertain additional ICC-IF location, while its main ICC-IF location is nucleoplasm and its tissue IHC profile is nuclear (HPA: subcellular summary; tissue IHC). | Judge paraffin IHC against the nuclear tissue pattern. Treat vesicular IF/ICC signal as uncertain supporting context, and investigate dominant cytoplasmic IHC staining with controls before assigning it to RFX1 (HPA: subcellular summary; general IHC practice). |
Experiment evidence
IHC & IF images showing RFX1 staining
A04392-1 · IHCIHC analysis of RFX1 using anti-RFX1 antibody (A04392-1). RFX1 was detected in a paraffin-embedded section of Diffuse large B-cell lymphoma
A04392-1 · IHCIHC analysis of RFX1 using anti-RFX1 antibody (A04392-1). RFX1 was detected in a paraffin-embedded section of human adrenocortical adenoma t
A04392-1 · IFIF analysis of RFX1 using anti-RFX1 antibody (A04392-1). RFX1 was detected in a paraffin-embedded section of human esophageal squamous cell
A04392 · IFImmunocytochemistry of RFX1 in HeLa cells with RFX1 at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132005-RFX1/tissue/cerebral+cortex#imid_13666923
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132005-RFX1/tissue/colon#imid_13666841
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132005-RFX1/tissue/liver#imid_13666842
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132005-RFX1/tissue/kidney#imid_13666851
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA048722; green = RFX1, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA048722; green = RFX1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RFX1 IHC & IF
🧪Run colon first: glandular cells should show nuclear RFX1 staining (HPA: High in colon glandular cells; UniProt P22670: Nucleus). Use prostate glandular cells as a negative tissue (HPA: Not detected); on the colon slide, use unstained non-glandular cells as an internal background comparison, without assuming every such cell is negative.
Positive control tissue: Colon (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RFX1 in MCF-7, PC-3, U2OS, HEK293, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control, and an RFX1 knockout sample or peptide-block control if a validated blocking peptide is available (standard IHC practice; caption: rabbit anti-RFX1 primary). Block endogenous peroxidase and inspect colon glandular lumens for nonspecific DAB signal; address endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the A04392-1 paraffin-section caption does not state a fixative. The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required or that these conditions are optimal. Check colon luminal deposits for staining artefacts (standard IHC practice); the supplied evidence does not establish whether frozen sections or IF are easier than paraffin-section IHC.
HPA tissue IHC evidence for RFX1
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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RFX1 IHC Tips
Troubleshoot RFX1 staining in paraffin section chromogenic IHC by checking nuclear localisation, the documented retrieval condition, and cell type matched controls.
Which retrieval condition should I try first for weak RFX1 staining?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04392-1). The selected tissue image used this condition, followed by 10% goat serum and 2 µg/mL catalog antibody overnight at 4°C (caption A04392-1). If nuclear staining remains weak, compare heating intervals on adjacent sections before trying citrate at pH 6.0 as a fallback; hold antibody and detection conditions constant (standard IHC practice). Prefer the condition that improves discrete nuclear signal without increasing diffuse background, since nuclear localisation is expected for RFX1 (UniProt P22670; HPA tissue IHC).
Could fixation explain weak or uneven RFX1 staining?
The selected paraffin section caption does not state a fixative, and target specific RFX1 sensitivity to fixation is unknown (caption A04392-1). Record the actual fixative, fixation duration and processing history for each specimen before comparing staining across sections (standard IHC practice). When specimens differ, test adjacent sections with the same EDTA pH 8.0 retrieval and 2 µg/mL antibody condition used in the selected image (datasheet A04392-1; caption A04392-1). Compare nuclear staining in intact cells and include matched controls; a difference between blocks alone cannot establish a target specific fixation effect (UniProt P22670; standard IHC practice).
How should I troubleshoot predominantly cytoplasmic RFX1 staining?
Expect nuclear RFX1 staining in chromogenic sections: UniProt assigns RFX1 to the nucleus, and HPA reports nuclear expression in most tissues (UniProt P22670; HPA tissue IHC). HPA places the main signal in the nucleoplasm, while its additional vesicle assignment is uncertain and comes from subcellular imaging (HPA subcellular). If DAB is predominantly cytoplasmic, compare adjacent sections with and without primary antibody, inspect nuclear counterstaining, and shorten DAB development if the precipitate obscures compartments (standard IHC practice). Score cells with clear nuclear signal separately from cytoplasmic signal, and avoid treating vesicle like staining alone as confirmation of RFX1 (HPA subcellular; standard IHC practice).
What can epitope information tell me when RFX1 staining varies?
The supplied record lists a single 1–979 chain and no annotated isoforms or transmembrane segment, so it provides no basis for assigning different tissue patterns to isoforms (UniProt P22670). It lists phosphoserines at positions 60, 978 and 979, but gives no antibody epitope or evidence that those modifications change IHC binding (UniProt P22670; caption A04392-1). If staining varies, first compare retrieval, processing and detection on matched sections, using the documented EDTA pH 8.0 condition as the starting point (datasheet A04392-1; standard IHC practice). Interpret any proposed epitope masking mechanism as untested until supported by antibody specific evidence (standard IHC practice).
How can IF help check an ambiguous chromogenic RFX1 result?
Use IF as a separate localisation check, since the chromogenic tissue caption documents paraffin section IHC rather than an IF workflow (caption A04392-1). For multiplex IF, pair RFX1 with a validated marker for the cell population being assessed and check whether its signal occupies nuclei within those marked cells (UniProt P22670; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence and include single stain controls to assess bleed through (standard IF practice). RFX1 has no transmembrane segment and is mainly nucleoplasmic, so optimise permeabilisation for access to the nuclear epitope while preserving morphology; the supplied evidence does not specify an IF permeabilisation condition for this antibody (UniProt P22670; HPA subcellular; caption A04392-1).
What should I change when RFX1 DAB staining looks diffuse?
Separate diffuse DAB deposition from discrete nuclear staining before changing the antibody concentration, because RFX1 is assigned to the nucleus (UniProt P22670). The selected image used 10% goat serum, 2 µg/mL primary antibody overnight at 4°C, and a peroxidase linked secondary incubated for 30 minutes at 37°C (caption A04392-1). Check a no primary control, endogenous peroxidase blocking, wash stringency and DAB development time on matched sections (standard IHC practice). If background persists, adjust one step at a time and retain a section showing credible nuclear signal as a comparison (HPA tissue IHC; standard IHC practice).
How should I quantify nuclear RFX1 across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then record the percentage of positive nuclei and nuclear intensity, or combine them in an H-score (standard IHC practice). Normalise positive counts to all evaluable nuclei in that cell population, or report positive nuclei per mm² of viable tissue when density is the endpoint (standard IHC practice). Keep retrieval, DAB development, counterstaining and intensity thresholds consistent across the comparison, and exclude folds and necrotic areas from scoring (standard IHC practice). Report cytoplasmic staining separately because UniProt and HPA support nuclear localisation, and note HPA's low staining versus RNA consistency when interpreting group differences (UniProt P22670; HPA tissue IHC).
How do I distinguish true RFX1 positivity from artefact?
A credible result shows defined nuclear staining in intact cells, consistent with UniProt nuclear localisation and HPA's nuclear tissue profile (UniProt P22670; HPA tissue IHC). As tissue references, HPA reports high staining in colon glandular cells and kidney tubular cells, but no detected staining in lymph node germinal center cells; compare the correct cell population rather than whole section colour (HPA tissue IHC). Inspect section edges, necrotic regions and no primary controls for nonspecific DAB or endogenous enzyme signal, and reject those areas when interpreting positivity (standard IHC practice). Treat agreement with HPA as supportive rather than decisive because its tissue IHC reliability note cites low consistency with RNA expression (HPA tissue IHC).
Boster reagents
Best RFX1 / MHC class II regulatory factor RFX1 IHC Antibodies
Catalog images show RFX1 IHC in human paraffin sections and IF/ICC in HeLa cells (A04392-1 IHC captions; A04392 IF captions). Both antibodies list human reactivity (catalog reactivity).
Real IHC data
Anti-RFX1 Antibody ®
Cat # A04392-1
Real IF data
Anti-RFX1 Antibody
Cat # A04392
A04392-1 lists human IHC and IF and shows staining in a paraffin-embedded section of human intestinal diffuse large B-cell lymphoma (catalog applications/reactivity; A04392-1 IHC caption). A04392 lists human IF and ICC and shows staining in HeLa cells (catalog applications/reactivity; A04392 IF captions).
Which to pick: Choose A04392-1 for tissue IHC: its own image shows paraffin-section staining, with the fixative unreported (A04392-1 IHC caption). Choose A04392 for IF/ICC in cells; its HeLa images support those applications, while clonality is unreported for both SKUs (A04392 IF captions; catalog clone fields). Neither SKU has stated reactivity beyond human, so no cross-species choice is supported (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P22670 (RFX1_HUMAN, MHC class II regulatory factor RFX1).
- Human Protein Atlas. RFX1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RFX1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. RFX1 antibody validation summary (2 antibodies).
- Study of FoxA pioneer factor at silent genes reveals Rfx-repressed enhancer at Cdx2 and a potential indicator of esophageal adenocarcinoma development. PLoS genetics 2011 — PMC3174211.
- Regulatory factor X1 promotes sorafenib-induced ferroptosis in hepatocellular carcinoma by transcriptional regulation of BECN1. Cellular oncology (Dordrecht, Netherlands) 2025 — PMC11996997.
- RFX1 Regulates Immune Microenvironment and Predicts Immunotherapy Response in Colon Cancer: A Multi-Omics and Clinical Analysis. Oncology research 2025 — PMC12712669.
- Dynamic changes in cis-regulatory occupancy by Six1 and its cooperative interactions with distinct cofactors drive lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium. Nucleic acids research 2020 — PMC7102962.
- PubMed PMID:2253877 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RFX1 (MHC class II regulatory factor RFX1). Expected localisation Nuclear staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04392-1). Validated IHC antibody: A04392-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Diffuse large B-cell lymphoma of human intestine tissue; fixative not specified…; 2. Fixation: Image fixative and duration unreported (datasheet A04392-1); 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 retrieval: EDTA pH 8.0 (datasheet A04392-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04392-1); 8. Primary antibody: Rabbit anti-RFX1, 2-5 μg/ml (datasheet A04392-1); 9. Primary incubation: Overnight at 4 °C (datasheet A04392-1). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: No isoforms or alternate processing annotated (UniProt); Regulation: Regulation not annotated (UniProt). Evidence: UniProt P22670, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A04392-1, PMC3174211, PMC11996997. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RFX1/immunohistochemistry-ihc-guide.