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CTCF / Transcriptional repressor CTCF · IHC design guide
Design Immunohistochemistry for CTCF
Plan CTCF IHC-P using the documented human colon example (datasheet M00132-1) and expected widespread nuclear staining (HPA tissue IHC). This guide covers fixation, positive tissue selection, and interpretation of low cholangiocyte staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CTCF 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 in tissue (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M00132-1) | |
| Positive control | Adrenal gland+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 | Formaldehyde-fixed paraffin sections are documented (selected-SKU IHC image M00132-1); keep fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Cholangiocytes show low staining (HPA tissue IHC) | |
| Regulation | Absent in primary spermatocytes (UniProt) | |
| Isoform / epitope | Two isoforms; epitope impact is unreported (UniProt) |
Section 1
Recommended CTCF IHC & IF Protocols
The catalog antibody protocol uses citrate pH 6 heat retrieval (datasheet M00132-1). Four published IHC protocols provide tissue-specific examples (PMC2409935; PMC10585925; PMC7229322; PMC6944131).
| Sample | Formaldehyde-fixed, paraffin-embedded human colon tissue (datasheet M00132-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet M00132-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet M00132-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CTCF, 1:25 (datasheet M00132-1) |
| Primary incubation | 1 hours at 37°C (datasheet M00132-1) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CTCF-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet M00132-1); adjust retrieval conditions if tissue staining needs optimization (standard IHC practice).
Section 2
What Is the Expected CTCF Staining Pattern?
CTCF is a nuclear protein with no transmembrane segment (UniProt P49711 topology and subcellular location). Expect staining in nuclei across many cell types: HPA reports ubiquitous nuclear expression and low tissue specificity, with tissue IHC reliability rated Supported because antibody staining is highly consistent with RNA expression (HPA tissue IHC). UniProt describes CTCF as ubiquitous but absent in primary spermatocytes (UniProt P49711 tissue specificity).
| Distinct nuclear staining in glandular, epithelial, myoepithelial, glial, or hematopoietic cells. | This fits the reported nuclear distribution (HPA tissue IHC; UniProt P49711 subcellular location). HPA reports High staining in adrenal and appendix glandular cells, bronchial respiratory epithelial cells, breast myoepithelial cells, cerebral cortex glial cells, and bone marrow hematopoietic cells (HPA tissue IHC). Compare nuclei with the local counterstain when scoring (general IHC practice). |
| Predominantly cytoplasmic or membrane staining, with little nuclear signal. | Question target attribution because nucleoplasm is the supported main location in ICC-IF and nuclear expression is the overall tissue IHC profile (HPA subcellular; HPA tissue IHC). Check the exception before rejecting a slide: HPA lists High cytoplasm/membrane staining in cerebellar molecular layer cells (HPA tissue IHC). |
| Strong staining in an unexpected cell population or acellular area. | Because CTCF is reported as ubiquitous, an unfamiliar positive cell type alone does not establish cross-reactivity (UniProt P49711 tissue specificity; HPA tissue IHC). Staining in primary spermatocytes conflicts with UniProt's reported absence; acellular color or signal outside cells warrants investigation for nonspecific detection or endogenous activity (UniProt P49711 tissue specificity; general IHC practice). |
| Haze or precipitate obscures nuclei throughout the section. | The result cannot be scored reliably for CTCF localization (general IHC practice). Diffuse color can arise from background or detection chemistry; assess a no-primary control and inspect whether staining follows tissue edges or debris (general IHC practice). HPA's Supported rating describes its reported staining pattern, not the performance of this run (HPA tissue IHC). |
| No nuclear signal in a tissue or cell population reported as High. | Treat this as a failed or indeterminate run until controls and detection are checked (general IHC practice). High reference examples include bone marrow hematopoietic cells and bronchial respiratory epithelial cells (HPA tissue IHC). Liver cholangiocytes are reported Low, so a faint result there alone is a weak failure indicator (HPA tissue IHC). |
💡Expected CTCF appearanceCall positive when cell nuclei show discernible chromogenic staining in an expected population, especially one reported High (HPA tissue IHC); predominant extranuclear color or diffuse section-wide color calls for review (HPA tissue IHC; general IHC practice).
| Reference tissue and cell population | HPA reports ubiquitous nuclear expression but different observed levels: several listed populations are High, while liver cholangiocytes are Low (HPA tissue IHC). Choose a reported High population as the positive reference and assess its nuclei; Low does not mean absent (HPA tissue IHC; general IHC practice). |
| Antibody evidence | HPA lists IHC as Supported for HPA004122, CAB062550, CAB068181, and CAB068182 (HPA antibodies). This supports their reported IHC patterns but does not validate every antibody, retrieval condition, or detection system (HPA antibodies; general IHC practice). |
| Cellular location and topology | UniProt places CTCF in the nucleoplasm and on chromosomes, including centromeres, and reports no transmembrane segment (UniProt P49711 subcellular location and topology). Score the nuclear compartment in routine tissue sections; avoid treating membrane color as the expected general pattern (HPA tissue IHC; UniProt P49711 topology). |
| Isoforms and processing | UniProt lists two isoforms and a 1–727 chain, with no signal peptide or propeptide reported (UniProt P49711 isoforms and processing). The supplied sources do not identify the catalog antibody's epitope or establish isoform-specific IHC behavior; interpret a divergent pattern using localization and controls rather than assigning it to an isoform (general IHC practice). |
| IF/ICC Q: Where should CTCF appear? | A: In the nucleoplasm; HPA calls this location supported and lists ICC-IF images for A-431, U-251MG, and U2OS (HPA subcellular). Use that as a localization cross-check for tissue IHC. It does not supply an IF protocol or establish identical staining intensity in paraffin sections (HPA subcellular; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear color in a reported High population. | The stain may have failed at retrieval, primary antibody, or detection steps; the observation alone cannot identify which step (general IHC practice). | Check the run's positive control and reagent records; verify that the catalog antibody is IHC-validated before adjusting one workflow variable at a time (HPA antibodies; general IHC practice). Use a reported High population as the reference (HPA tissue IHC). |
| Only faint staining in liver cholangiocytes. | HPA reports Low staining in these cells, so faint color may match the reference (HPA tissue IHC). | Compare with nuclei in a reported High population on an appropriate control section before calling the run unsuccessful (HPA tissue IHC; general IHC practice). |
| Strong cytoplasmic color with weak nuclei. | This differs from the general nuclear profile, although HPA reports High cytoplasm/membrane staining for cerebellar molecular layer cells (HPA tissue IHC). | Identify the tissue and cell population, compare nuclear counterstaining, and review a no-primary control; investigate broad extranuclear staining outside the reported exception (HPA tissue IHC; general IHC practice). |
| Uniform brown color or granular deposit across tissue and empty areas. | Background deposition or endogenous detection activity can obscure the specific nuclear pattern (general IHC practice). | Inspect no-primary and detection controls; review blocking, washes, and chromogen development according to the chosen detection system (general IHC practice). Do not score diffuse color as nuclear CTCF (HPA tissue IHC). |
| Positive staining appears in primary spermatocytes. | UniProt reports CTCF absent in primary spermatocytes, so this result conflicts with that reference; identity or specificity remains unresolved (UniProt P49711 tissue specificity). | Confirm the cell identification and nuclear localization, then compare appropriate control sections and antibody evidence before interpreting the signal as CTCF (general IHC practice; HPA antibodies). |
| Different antibodies give different compartments. | HPA's Supported IHC entries do not establish that every antibody and assay condition yields the same image (HPA antibodies; general IHC practice). | Compare each antibody's validated application and controls, then prioritize a reproducible nuclear result consistent with the tissue profile; document any cerebellar molecular layer exception separately (HPA antibodies; HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CTCF staining
M00132-1 · IHCM00132-1 staining CTCF in human colon tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). T
M00132-3 · IHCMouse kidney was stained with anti-CTCF rabbit antibody
M00132-3 · IHCRat kidney was stained with anti-CTCF rabbit antibody
M00132 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
PB9493 · IFIF analysis of CTCF using anti-CTCF antibody (PB9493) and anti-Tubulin Alpha antibody (M03989-3). CTCF was detected in immunocytochemical se
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000102974-CTCF/tissue/cerebral+cortex#imid_18885269
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000102974-CTCF/tissue/colon#imid_18885195
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000102974-CTCF/tissue/liver#imid_18885196
HPA · GallbladderHuman Protein Atlas IHC, Gallbladder. Source: https://www.proteinatlas.org/ENSG00000102974-CTCF/tissue/gallbladder#imid_18885188
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004122; green = CTCF, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004122; green = CTCF, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CTCF IHC & IF
🧪Run bronchus first: respiratory epithelial cells should show nuclear CTCF staining (HPA: High in respiratory epithelial cells; UniProt P49711: nucleus). HPA detects CTCF in all 45 scored tissues, so there is no validated negative tissue or reliably negative cell population within the positive slide; nuclei without signal should be interpreted against the no-primary and isotype controls (HPA: no negative rows; UniProt P49711: ubiquitous).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CTCF 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 CTCF in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a control antibody matched to the primary’s host species, isotype and clonality; use CTCF knockout material as a biological specificity control if available (standard IHC practice). For chromogenic bronchus IHC, block endogenous peroxidase and inspect inflammatory cells for background staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected M00132-1 tissue-IHC caption’s fixative field is unreported (selected-SKU caption: fixative not stated). The caption uses heat-mediated citrate retrieval at pH 6, but does not establish that retrieval is required for CTCF (selected-SKU caption: citrate retrieval). Frozen-section ease is unreported; IF localisation is supported in the nucleoplasm, while bronchial mucus and inflammatory cells can complicate background assessment (HPA: nucleoplasm supported; standard IHC/IF practice).
HPA tissue IHC evidence for CTCF
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CTCF is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CTCF IHC Tips
Troubleshoot CTCF staining in paraffin sections by checking retrieval, nuclear localisation, controls and scoring before interpreting differences between samples.
How should I retrieve CTCF in paraffin sections when nuclear staining is weak?
Start with heat-mediated citrate antigen retrieval at pH 6 for this catalog antibody (datasheet M00132-1). In the selected tissue IHC example, that retrieval preceded a 1:25 primary antibody incubation for 1 hour at 37°C (M00132-1 tissue IHC caption). Compare weak and strong areas within the same section, then adjust heating duration in a small pilot while keeping the antibody dilution and detection steps fixed (general IHC practice). Score nuclear signal separately from diffuse staining, since CTCF is principally nucleoplasmic (HPA: nucleoplasm supported; UniProt P49711 subcellular location).
Could fixation explain variable CTCF staining across paraffin blocks?
CTCF-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; treat fixation as a variable to test, not an established cause. Compare sections with documented processing histories in the same staining run, using the same pH 6 citrate retrieval and primary antibody conditions (datasheet M00132-1; general IHC practice). Examine intact regions away from folds, edges and necrosis before attributing a weak nucleus to antigen loss (general IHC practice). Record whether nuclear signal and tissue morphology change together across blocks, and confirm any proposed fixation effect with matched specimens processed under controlled conditions (general IHC practice).
Should CTCF staining be nuclear in every cell and cell cycle stage?
Expect predominantly nuclear staining in paraffin sections: tissue IHC shows ubiquitous nuclear expression, and the supported subcellular location is nucleoplasm (HPA: tissue IHC profile; HPA: nucleoplasm supported). Interpret occasional mitotic patterns cautiously because CTCF associates with centromeres and chromosomal arms during metaphase (UniProt P49711 subcellular location). Nucleolar redistribution may occur upon differentiation, so assess morphology and neighbouring nuclei before calling an unusual nuclear pattern nonspecific (UniProt P49711 subcellular location; general IHC practice). Widespread cytoplasmic staining without convincing nuclei warrants a detection and background check; CTCF has no annotated transmembrane segment (UniProt P49711 topology).
Can this IHC stain distinguish CTCF isoforms or modification states?
Do not assign an IHC intensity difference to a particular CTCF isoform without knowing the antibody epitope and validating isoform selectivity (general IHC practice). The record lists 2 isoforms, 1 and 2, while the supplied IHC caption gives no epitope or isoform discrimination data (UniProt P49711 isoforms; M00132-1 tissue IHC caption). Several modified residues are annotated, including phosphothreonine at 289 and phosphoserine at 402 (UniProt P49711 modified residues). If modification sensitivity matters, compare an independently validated modification-specific assay with total CTCF staining; nuclear IHC alone cannot establish modification state (general IHC practice).
How can IF help resolve ambiguous CTCF-positive cells in an IHC section?
On a comparable section, multiplex CTCF IF with a marker identifying the expected glandular cell population; appendix glandular cells show high tissue IHC expression (HPA: High in appendix glandular cells; general IF practice). Include a nuclear counterstain and inspect whether the CTCF channel overlaps nuclei, consistent with its supported nucleoplasmic location (HPA: nucleoplasm supported; general IF practice). Choose a red or far-red fluorophore after checking tissue autofluorescence and single-channel controls (general IF practice). Because CTCF is nuclear and has no transmembrane segment, optimise permeabilisation for nuclear access rather than for an extracellular epitope (UniProt P49711 subcellular location and topology; general IF practice).
How do I distinguish CTCF signal from chromogenic background?
First inspect a no-primary control and separate crisp nuclear staining from diffuse cytoplasmic or stromal colour, since CTCF is predominantly nucleoplasmic (HPA: nucleoplasm supported; general IHC practice). The selected example used 3% BSA blocking for 0.5 hour and a biotinylated secondary antibody (M00132-1 tissue IHC caption). If background persists, review blocking, washing and detection exposure one variable at a time, including possible endogenous biotin where a biotin-based system is used (general IHC practice). Apply a peroxidase block when using enzyme-based DAB detection, and check whether residual colour appears in the no-primary control (general IHC practice).
What is a defensible way to quantify CTCF IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define intact, comparable regions before scoring and count only nuclei that meet a prespecified staining threshold (general IHC practice; HPA: ubiquitous nuclear expression). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score calculated from intensity grades 0–3 on a 0–300 scale (general IHC practice). Normalise positive counts to all evaluable nuclei in the same defined cell population; if reporting positive-cell density, state the sampled area in mm² (general IHC practice). Keep retrieval, detection and image settings consistent, and exclude folds, edges and necrotic regions by the same rules for every section (general IHC practice).
When does an unusual CTCF staining pattern indicate artefact?
Treat crisp nuclear staining as the main expected pattern, supported by ubiquitous nuclear tissue expression and nucleoplasmic localisation (HPA: tissue IHC profile; HPA: nucleoplasm supported). Interpret an apparent negative population against tissue context: cholangiocytes have low reported staining, while appendix glandular cells have high staining (HPA: Low in liver cholangiocytes; HPA: High in appendix glandular cells). Diffuse cytoplasmic colour, staining confined to section edges or necrosis, and colour in a no-primary control favour artefact over a CTCF localisation claim (general IHC practice). Check endogenous enzyme activity in chromogenic detection before assigning unusual colour to target expression (general IHC practice).
Boster reagents
Best CTCF / Transcriptional repressor CTCF IHC Antibodies
Anti-CTCF antibodies have IHC images from human colon and mouse and rat kidney, plus IF images from U2OS cells and an unspecified specimen (catalog image captions).
Real IHC data
Anti-CTCF Antibody (Center)
Cat # M00132-1
Real IHC data
Anti-CTCF Rabbit Monoclonal Antibody
Cat # M00132-3
Real IF data
Anti-CTCF Rabbit Monoclonal Antibody
Cat # M00132
Real IF data
Anti-CTCF Antibody ®
Cat # PB9493
M00132-1 shows human colon paraffin-section IHC (M00132-1 image caption); M00132-3 shows mouse and rat kidney IHC (M00132-3 image captions). M00132 has an IF image without an identified specimen and lists IHC, ICC and IF applications (M00132 image caption; catalog applications); PB9493 shows IF in U2OS cells (PB9493 image caption).
Which to pick: For human paraffin-section IHC, start with M00132-1 at 1:25; its colon caption reports formaldehyde fixation, citrate pH 6 heat retrieval and a biotinylated secondary antibody (M00132-1 image caption; catalog IHC dilution). For IF/ICC, PB9493 has a U2OS cell IF image at 5 μg/ml, while M00132 is a monoclonal with an IF image and listed ICC/IF applications (PB9493 image caption; M00132 catalog clone/applications and image caption). For cross-species tissue IHC, M00132-3 is a rabbit monoclonal listed for human, mouse and rat, with mouse and rat kidney images; its image captions do not report fixation or paraffin processing (M00132-3 catalog title/reactivity/applications; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49711 (CTCF_HUMAN, Transcriptional repressor CTCF).
- Human Protein Atlas. CTCF tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CTCF subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. CTCF antibody validation summary (4 antibodies).
- Expression of the transcription factor CTCF in invasive breast cancer: a candidate gene located at 16q22.1. British journal of cancer 2004 — PMC2409935.
- Transgenerational inheritance of adrenal steroidogenesis inhibition induced by prenatal dexamethasone exposure and its intrauterine mechanism. Cell communication and signaling : CCS 2023 — PMC10585925.
- Association of mutation and low expression of the CTCF gene with breast cancer progression. Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society 2020 — PMC7229322.
- Expression of CCCTC-binding factor (CTCF) is linked to poor prognosis in prostate cancer. Molecular oncology 2020 — PMC6944131.
- PubMed PMID:8649389 — UniProt-cited evidence.
- PubMed PMID:9591631 — UniProt-cited evidence.
- PubMed PMID:11782357 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CTCF (Transcriptional repressor CTCF). Expected localisation Nuclear in tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M00132-1). Validated IHC antibody: M00132-1 at 1:25 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded human colon tissue (datasheet M00132-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet M00132-1); 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: Citrate pH 6 (datasheet M00132-1); 20 min, 95–100 °C (standard); 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-CTCF, 1:25 (datasheet M00132-1); 9. Primary incubation: 1 hours at 37°C (datasheet M00132-1). Watch for: Fixation: Formaldehyde-fixed paraffin sections are documented (selected-SKU IHC image M00132-1); keep fixation consistent. (standard IHC practice; not target-specific); Caveat: Cholangiocytes show low staining (HPA tissue IHC); Isoform / epitope: Two isoforms; epitope impact is unreported (UniProt); Regulation: Absent in primary spermatocytes (UniProt). Evidence: UniProt P49711, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet M00132-1, PMC2409935, PMC10585925. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CTCF/immunohistochemistry-ihc-guide.