This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CDX2 / Homeobox protein CDX-2 · IHC design guide
Design Immunohistochemistry for CDX2
Plan CDX2 staining in paraffin sections around the nuclear intestinal pattern reported by HPA (HPA tissue IHC). Use intestinal tissue as a positive reference and compare nuclear staining across consistently fixed sections (HPA tissue IHC; standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CDX2 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 intestinal tract (HPA tissue IHC) | |
| Staining pattern | Intestinal endocrine cell nuclei stain (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M00877-1) | |
| Positive control | Appendix+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 consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Nonintestinal tissues may lack detectable staining (HPA tissue IHC) | |
| Regulation | Expression regulation is not specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–313 chain (UniProt) |
Section 1
Recommended CDX2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses heat retrieval with EDTA at pH 8.0 (datasheet: M00877-1). The published MPNST protocol below reports its retrieval and primary dilution (PMC9097546).
| Sample | Paraffin-embedded human colon tissue; fixative not specified (datasheet M00877-1) |
| Fixation | Image fixative and duration unreported (datasheet M00877-1); 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 M00877-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00877-1) |
| Primary antibody | Rabbit monoclonal (clone AFA-3) anti-CDX2, 1:50 (datasheet M00877-1) |
| Primary incubation | Overnight at 4 °C (datasheet M00877-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M00877-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CDX2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in intestinal tract. No signal in the no-primary control. |
💡Decision noteStart with EDTA at pH 8.0 for the catalog antibody (datasheet: M00877-1). Use the published retrieval reagent when reproducing the MPNST protocol (PMC9097546).
Section 2
What Is the Expected CDX2 Staining Pattern?
CDX2 should appear predominantly in nuclei in intestinal tract sections, with high staining reported in endocrine cells of the appendix, colon, duodenum and rectum, and medium staining in small-intestinal endocrine cells (HPA tissue IHC). HPA rates the tissue pattern Enhanced, citing consistency between staining and RNA expression (HPA tissue IHC). Nuclear localisation fits CDX2’s transcription-factor role and lack of a transmembrane segment (UniProt Q99626).
| Distinct nuclear staining in intestinal endocrine cells, with little surrounding background. | This matches the reported nuclear intestinal-tract pattern. Compare intensity within the sampled tissue: HPA reports High in appendix, colon, duodenum and rectum endocrine cells, and Medium in small-intestinal endocrine cells (HPA tissue IHC). Score the stained cells and compartment, rather than calling an entire tissue positive from a few nuclei. |
| Predominantly cytoplasmic staining with no convincing nuclear signal. | Treat this as an uncertain IHC result: the expected tissue pattern is nuclear (HPA tissue IHC; UniProt Q99626). HPA also reports cytosol and vesicles as additional ICC-IF locations, so cytoplasmic signal alone does not prove artefact (HPA subcellular ICC-IF). Recheck nuclear localisation in a positive control before assigning the slide a CDX2-positive score. |
| Strong staining in a cell type reported as negative, such as bronchial respiratory epithelium. | HPA reports CDX2 as Not detected in bronchial respiratory epithelial cells (HPA tissue IHC). Unexpected staining raises possible antibody cross-reactivity or endogenous detection activity; it does not identify the cause by itself. Compare the same run’s positive tissue and appropriate detection controls before interpreting that signal as CDX2. |
| Diffuse colour across cells and surrounding tissue obscures nuclear boundaries. | This is background rather than a scorable nuclear pattern. In a chromogenic IHC workflow, assess blocking, wash steps and detection controls as general practice; HPA does not attribute CDX2 staining to any specific background mechanism. Interpret only nuclei that remain clearly distinguishable from surrounding colour. |
| No visible nuclear staining in endocrine cells of an otherwise suitable colon section. | HPA reports High staining in colon endocrine cells (HPA tissue IHC), so an absent signal calls for a run-level check before a biological-negative conclusion. Confirm the cells are present, inspect the positive control, then review the antibody and detection workflow. HPA’s tissue pattern alone cannot identify which step failed. |
💡Expected CDX2 appearanceA convincing positive is predominantly nuclear colour in intestinal endocrine cells, typically High in colon and Medium in small intestine; isolated diffuse or wrong-cell staining is suspect rather than sufficient evidence of CDX2 (HPA tissue IHC).
| Tissue and cell selection | Use a reported positive cell population when judging the run: colon endocrine cells are High, while small-intestinal endocrine cells are Medium (HPA tissue IHC). HPA also lists adipocytes in adipose tissue and bronchial respiratory epithelial cells as Not detected; these are context for interpretation, not proof that every cell in those sections is negative (HPA tissue IHC). |
| IHC antibody evidence | CAB002221 is listed as IHC Enhanced (HPA antibodies). HPA’s overall tissue reliability is also Enhanced, with staining consistent with RNA expression (HPA tissue IHC). These labels support the reported pattern; they do not establish that another antibody or a changed workflow will reproduce it. |
| Cellular localisation | UniProt places CDX2 in the nucleus and lists no transmembrane segment (UniProt Q99626). Nuclear signal should therefore anchor IHC scoring. HPA’s additional cytosol and vesicle locations come from ICC-IF and should be kept distinct from its nuclear tissue-IHC profile (HPA subcellular ICC-IF; HPA tissue IHC). |
| IF/ICC: what pattern should be expected? | The main reported location is nucleoplasm, with additional vesicle and cytosol localisation (HPA subcellular ICC-IF). This is an interpretation cue for IF/ICC images, not an IHC protocol option. HPA lists CACO-2, HEK293, U2OS, JURKAT and NIH 3T3 as lines with ICC-IF images; that listing does not establish equal staining in every line (HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive intestinal control lacks nuclear colour. | A failed staining or detection step is possible; the slide alone does not locate the failure. | Verify the reported positive cells are present, then check primary antibody use, chromogen development and run controls as general IHC practice. Colon endocrine cells are reported High (HPA tissue IHC); do not score a test section negative until the control works. |
| Only cytoplasmic colour is apparent in a test section. | The result does not match the dominant nuclear tissue pattern (HPA tissue IHC). | Compare with a concurrently stained intestinal positive section and inspect nuclei at suitable magnification. Record any cytoplasmic signal separately: HPA reports additional cytosol and vesicle localisation in ICC-IF, which does not by itself validate cytoplasm-only IHC (HPA subcellular ICC-IF). |
| Unexpected colour appears in bronchial respiratory epithelial cells. | Cross-reactivity or endogenous detection activity is possible; HPA reports those cells as Not detected (HPA tissue IHC). | Check a detection-only or no-primary control as general IHC practice, and compare the staining compartment with a positive intestinal control. If the unexpected pattern persists, treat the result as unresolved rather than calling it CDX2 expression. |
| Widespread haze prevents confident nuclear scoring. | Non-specific background in staining or detection is possible; the cause is not established by HPA. | Review blocking, wash and chromogen steps, and inspect the negative control as general chromogenic IHC practice. Repeat the run if nuclear boundaries cannot be distinguished; a broad colour wash cannot support a compartment-specific CDX2 call. |
| Small-intestinal endocrine cells stain less strongly than colon endocrine cells. | This difference can match HPA’s reported Medium versus High levels (HPA tissue IHC). | Assess localisation and the controls before changing conditions. Do not require colon-level intensity in small intestine; compare like cell populations under the same scoring approach (HPA tissue IHC). |
| A pancreas section gives an unexpected result. | UniProt reports CDX2 detection in pancreas, but the supplied HPA tissue-IHC positives do not give a pancreatic cell-level staining pattern (UniProt Q99626; HPA tissue IHC). | Avoid assigning an expected pancreatic intensity or cell type from this evidence. Interpret the section with a verified intestinal positive control and report the pancreatic observation separately from the HPA-supported pattern. |
Experiment evidence
IHC & IF images showing CDX2 staining
M00877-1 · IHCImmunohistochemical analysis of paraffin-embedded human colon, using CDX2 Antibody.
M00877-1 · IHCIHC analysis of CDX2 using anti-CDX2 antibody (M00877-1). CDX2 was detected in a paraffin-embedded section of human colon cancer tissue. Hea
A00877-1 · IHCImmunohistochemistry of CDX2 in rat brain tissue with CDX2 antibody at 5 μg/mL.
A00877-1 · IFImmunofluorescence of CDX2 in rat brain tissue with CDX2 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165556-CDX2/tissue/cerebral+cortex#imid_710544
HPA · DuodenumHuman Protein Atlas IHC, Duodenum. Source: https://www.proteinatlas.org/ENSG00000165556-CDX2/tissue/duodenum#imid_710485
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165556-CDX2/tissue/colon#imid_710478
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165556-CDX2/tissue/liver#imid_710479
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA045669; green = CDX2, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA045669; green = CDX2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CDX2 IHC & IF
🧪Run colon first and look for nuclear staining in endocrine cells (HPA: High in colon endocrine cells; UniProt Q99626: nucleus). Use bronchus respiratory epithelium as the negative tissue (HPA: Not detected in bronchus respiratory epithelial cells); on the colon slide, neighboring stromal nuclei should provide an unstained internal reference (standard IHC practice).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CDX2 in CACO-2, HEK293, U2OS, JURKAT, NIH 3T3, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary secondary-only control, an isotype control matched to the primary antibody’s host species and monoclonal or polyclonal format, and verified CDX2-knockout material or a validated peptide-block control (standard IHC practice). For colon chromogenic IHC, quench endogenous peroxidase and check for background in the no-primary control (standard IHC practice).
⚠️Feasibility: A CDX2-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected M00877-1 paraffin-section colon caption does not state a fixative (selected-SKU caption: fixative not stated). Antigen retrieval may be needed for paraffin IHC, but CDX2-specific retrieval dependence is unreported; the supplied evidence does not establish whether frozen sections or IF are easier (standard IHC practice; supplied evidence). In colon, assess crisp nuclear staining separately from luminal mucus or deposits that can complicate interpretation (UniProt Q99626: nucleus; standard IHC practice).
HPA tissue IHC evidence for CDX2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Rectum | Endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | Endocrine 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CDX2 IHC Tips
Troubleshoot CDX2 staining by checking nuclear signal, intestinal tissue controls, and workflow controls before interpreting chromogenic IHC results.
How should I retrieve CDX2 antigen when nuclear staining is weak?
Start with heat-mediated EDTA pH 8.0 antigen retrieval for the catalog antibody (datasheet M00877-1). If nuclear staining remains weak, compare a 10–20 minute heating range while keeping section thickness, cooling, and detection conditions consistent (standard IHC practice). Include a colon section as a tissue control: the selected product has a paraffin-section colon IHC image, although its caption does not report the fixative (caption M00877-1). Score improvement in nuclei rather than diffuse color, because CDX2 is nuclear (UniProt Q99626) and intestinal-tract expression is predominantly nuclear (HPA tissue IHC).
Could fixation explain weak CDX2 staining in my paraffin sections?
Target-specific CDX2 sensitivity to fixation is unknown from the supplied evidence; the selected colon IHC caption reports paraffin embedding but gives no fixative (caption M00877-1). Record the actual fixative, fixation duration, processing schedule, and section age for each case before comparing staining (standard IHC practice). If fixation varies, stain representative sections together using EDTA pH 8.0 retrieval and the same detection run (datasheet M00877-1; standard IHC practice). A weak result should be judged against an intestinal tissue control and section quality, without assigning a CDX2-specific fixation effect that has not been demonstrated (HPA tissue IHC; standard IHC practice).
Which staining compartment should count as a CDX2-positive IHC result?
Score crisp nuclear staining in morphologically intact cells as the primary CDX2 result, because CDX2 is a nuclear transcription factor (UniProt Q99626). The tissue profile describes nuclear expression in the intestinal tract, and colon endocrine cells show high staining (HPA tissue IHC). Cytoplasmic color alone should prompt review of background and morphology; subcellular IF data also report cytosol and vesicles, so compartment assignment needs assay context (HPA subcellular). Compare nuclei with an appropriate hematoxylin counterstain and inspect a control section before setting a positivity threshold (standard IHC practice). Keep nuclear and nonnuclear observations separate in the report (standard IHC practice).
Could an isoform or modified epitope cause inconsistent CDX2 staining?
The supplied record annotates 0 isoforms and one 1–313 amino-acid chain, so it offers no specific isoform explanation for discordant IHC (UniProt Q99626). Phosphoserines at residues 60 and 283 are annotated, but their effect on this antibody is unknown because its epitope is not supplied (UniProt Q99626; supplied antibody evidence). Check the antibody’s epitope documentation before attributing a loss of nuclear signal to modification or truncation (standard IHC practice). Compare staining across intestinal control sections processed in the same run, then investigate retrieval and detection variables if results diverge (HPA tissue IHC; standard IHC practice).
How can I check CDX2 by IF alongside the chromogenic IHC result?
Use IF as a separate localisation check and pair CDX2 with a validated marker of the expected intestinal cell type; the supplied tissue profile identifies intestinal endocrine cells among positive populations (HPA tissue IHC; standard IF practice). Select a fluorophore whose signal is well separated from the section’s measured autofluorescence, and acquire single-label controls before interpreting overlap (standard IF practice). CDX2 has no transmembrane segment and its principal location is nuclear, so choose permeabilisation that permits antibody access to nuclear antigen (UniProt Q99626; standard IF practice). Do not transfer the paraffin colon caption’s unstated fixation conditions into the IF setup (caption M00877-1).
How do I distinguish background color from genuine CDX2 staining?
Genuine chromogenic signal should be evaluated in nuclei against tissue morphology, since CDX2 is nuclear and the tissue profile shows intestinal-tract nuclear expression (UniProt Q99626; HPA tissue IHC). For a peroxidase and DAB workflow, include an endogenous-peroxidase block and a primary-antibody omission control to reveal enzyme-derived or detection background (standard IHC practice). Review diffuse cytoplasmic color, precipitate, and staining concentrated at section edges before calling cells positive (standard IHC practice). If background persists, compare blocking and washing conditions while keeping retrieval at EDTA pH 8.0 and checking whether the nuclear control remains visible (datasheet M00877-1; standard IHC practice).
What is a defensible way to quantify CDX2 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and count only interpretable nuclei, consistent with CDX2’s nuclear localisation (UniProt Q99626; standard IHC practice). Report the percentage of nuclear-positive cells and, when intensity is reproducible, an H-score using intensity categories 0–3 and a total range of 0–300 (standard IHC practice). Normalise positive counts to the number of evaluable cells in the same annotated tissue area; if using density, report positive nuclei per mm² of viable tissue (standard IHC practice). Apply one prespecified threshold, exclude damaged regions, and compare sections stained in the same run with an intestinal control (HPA tissue IHC; standard IHC practice).
When is an unexpected CDX2-positive IHC result credible?
A credible result has cell-associated nuclear signal in viable tissue, matching CDX2’s nuclear localisation and the intestinal-tract tissue profile (UniProt Q99626; HPA tissue IHC). Check the stained cell type: high signal is reported in colon, appendix, duodenum, and rectum endocrine cells, whereas several listed nonintestinal populations were not detected (HPA tissue IHC). Recheck isolated staining at cut edges or in necrotic areas, and exclude endogenous-enzyme color with appropriate controls before calling an unexpected cell positive (standard IHC practice). Interpret any discordance alongside morphology and control performance; a positive DAB deposit alone does not establish CDX2 expression (standard IHC practice).
Boster reagents
Best CDX2 / Homeobox protein CDX-2 IHC Antibodies
Anti-CDX2 antibodies have IHC images from paraffin-embedded human and mouse colon and from rat brain; rat brain also has an IF image (catalog image captions).
Real IHC data
Anti-CDX2 Rabbit Monoclonal Antibody
Cat # M00877-1
Real IHC data
Anti-Homeobox protein CDX-2 CDX2 Antibody
Cat # A00877-1
M00877-1 is listed for IHC and ICC/IF in human, mouse, and rat, with IHC images from paraffin-embedded human and mouse colon (catalog applications, reactivity, and IHC captions). A00877-1 is listed for IHC-P and IF in human, mouse, and rat, with IHC and IF images from rat brain (catalog applications, reactivity, and image captions).
Which to pick: For colon tissue IHC, choose the rabbit monoclonal M00877-1: its captions document paraffin sections of human and mouse colon at 1:50; the fixative is unreported (catalog host, clone, dilution, and IHC captions). For IF, choose A00877-1 when a tissue IF image matters: its rat-brain caption shows IF at 20 μg/mL; M00877-1 lists ICC/IF but has no IF image in this payload (catalog applications and IF captions). For cross-species work, both list human, mouse, and rat reactivity, with IHC images covering human and mouse for M00877-1 and rat for A00877-1; A00877-1’s IHC caption does not report tissue processing or fixative (catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q99626 (CDX2_HUMAN, Homeobox protein CDX-2).
- Human Protein Atlas. CDX2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CDX2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles and cytosol..
- Human Protein Atlas. CDX2 antibody validation summary (3 antibodies).
- Evaluation of the Immunohistochemical Scoring System of CDX2 Expression as a Prognostic Biomarker in Colon Cancer. Diagnostics (Basel, Switzerland) 2024 — PMC11119288.
- Special AT-rich sequence binding protein 2 (SATB2), β-catenin, and CD10 immunohistochemistry provides insights into the histopathologic features and cellular differentiation of adenomatoid odontogenic tumor. Journal of dental sciences 2026 — PMC13535953.
- CDX2 expression in malignant peripheral nerve sheath tumour: a potential diagnostic pitfall associated with PRC2 inactivation. Histopathology 2022 — PMC9097546.
- Diagnostic and Prognostic Roles of CDX2 Immunohistochemical Expression in Colorectal Cancers. Diagnostics (Basel, Switzerland) 2022 — PMC8947721.
- PubMed PMID:9459001 — UniProt-cited evidence.
- PubMed PMID:9036867 — UniProt-cited evidence.
- PubMed PMID:11161380 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CDX2 (Homeobox protein CDX-2). Expected localisation Nuclear staining in intestinal tract (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M00877-1). Validated IHC antibody: M00877-1 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon tissue; fixative not specified (datasheet M00877-1); 2. Fixation: Image fixative and duration unreported (datasheet M00877-1); 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 M00877-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00877-1); 8. Primary antibody: Rabbit monoclonal (clone AFA-3) anti-CDX2, 1:50 (datasheet M00877-1); 9. Primary incubation: Overnight at 4 °C (datasheet M00877-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Nonintestinal tissues may lack detectable staining (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–313 chain (UniProt); Regulation: Expression regulation is not specified (UniProt). Evidence: UniProt Q99626, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet M00877-1, PMC9097546. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CDX2/immunohistochemistry-ihc-guide.