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ANXA6 / Annexin A6 · IHC design guide
Design Immunohistochemistry for ANXA6
Plan ANXA6 staining in paraffin sections using its widespread cytoplasmic tissue pattern as a starting point (HPA tissue IHC). Compare cell populations within each section because detectable signal varies by cell type (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ANXA6 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 (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03735-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Esophagus+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 | Some cell populations have no detectable signal (HPA tissue IHC) | |
| Regulation | Low tissue specificity; no regulator annotated (HPA tissue IHC; UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended ANXA6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M03735-1) is followed by published ANXA6 protocols for rat abdominal aorta (PMC9492406) and human breast tumor cores (PMC8266800).
| Sample | Paraffin-embedded human liver tissue; fixative not specified (datasheet M03735-1) |
| Fixation | Image fixative and duration unreported (datasheet M03735-1); 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 M03735-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03735-1) |
| Primary antibody | Mouse monoclonal (clone 4C4) anti-ANXA6, 2 μg/ml (datasheet M03735-1) |
| Primary incubation | Overnight at 4 °C (datasheet M03735-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M03735-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ANXA6-positive staining in glandular cells of adrenal gland (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 (datasheet: M03735-1); neither published IHC excerpt specifies retrieval conditions (PMC9492406; PMC8266800).
Section 2
What Is the Expected ANXA6 Staining Pattern?
ANXA6 should appear predominantly in the cytoplasm of stained cells (HPA: ubiquitous cytoplasmic expression; UniProt P08133: cytoplasm). Strong examples include adrenal glandular cells, lung alveolar type II cells, and pancreatic endocrine cells (HPA: High in each). ANXA6 has no transmembrane segment (UniProt P08133 topology). HPA rates its tissue staining reliability Enhanced, based on agreement with RNA expression, pending external verification (HPA: reliability description).
| Cytoplasmic chromogen in adrenal glandular cells, lung alveolar type II cells, or pancreatic endocrine cells (HPA: High in each). | This matches the expected compartment and cell types (HPA: ubiquitous cytoplasmic expression; HPA: High in each). Assess each cell population separately: a strong positive population does not make every nearby cell positive. |
| Predominantly nuclear staining, with little or no cytoplasmic staining. | A nuclear-only result conflicts with the reported cytoplasmic pattern (HPA: tissue IHC profile; UniProt P08133: cytoplasm). Treat it as suspect and check antibody specificity, detection background, and the staining run before scoring it as ANXA6. |
| Strong staining in esophageal squamous epithelial cells or gallbladder glandular cells (HPA: Not detected in each). | That differs from the reported staining for those specific cell types (HPA: Not detected in each). Check for cross-reactivity or endogenous detection activity; do not infer that the entire tissue must be negative (standard IHC practice). |
| Diffuse chromogen across tissue, extracellular space, or the slide, without clear cell boundaries. | This obscures the expected cytoplasmic pattern (HPA: tissue IHC profile). Review blocking, washes, detection reagents, and a control processed without primary antibody to identify nonspecific background (standard IHC practice). |
| No signal in a section containing adrenal glandular cells previously expected to stain (HPA: High). | A negative run in an HPA-reported high population warrants a technical check, but a single section cannot establish ANXA6 absence. Confirm tissue identity and assay controls, then review retrieval, antibody dilution, and detection (standard IHC practice). |
💡Expected ANXA6 appearanceA convincing positive result is cell-associated cytoplasmic staining in an HPA-reported High population, such as adrenal glandular cells; dominant nuclear staining or diffuse cell-free chromogen is suspect (HPA: tissue IHC profile; HPA: High in adrenal glandular cells; UniProt P08133: cytoplasm).
| Cell population and tissue | HPA describes ubiquitous cytoplasmic expression but low tissue RNA specificity; IHC intensity still varies by cell type (HPA: tissue IHC profile; HPA: RNA specificity). Its Not detected calls apply to the named populations, including gallbladder glandular cells, rather than every cell in those tissues (HPA: tissue IHC). |
| Compartment and topology | Interpret a cytoplasmic pattern as the main tissue IHC expectation (HPA: tissue IHC profile; UniProt P08133: cytoplasm). ANXA6 has no transmembrane segment, so membrane-only staining needs corroboration before assignment to ANXA6 (UniProt P08133 topology). |
| IHC evidence strength | HPA rates the overall tissue profile Enhanced for staining–RNA agreement, while external verification is pending (HPA: reliability description). Antibodies HPA002462 and HPA009650 each have Enhanced IHC status; CAB005077 is Supported (HPA: antibody validation). |
| What should IF/ICC show? | The separate IF/ICC evidence reports mainly cytosolic signal, with an additional basal-body location; both locations are Approved (HPA: subcellular ICC-IF). That observation should not be imposed as a required basal-body pattern in chromogenic tissue IHC (HPA: subcellular ICC-IF; HPA: tissue IHC profile). |
| Fixation and retrieval evidence | The supplied HPA and UniProt records report no ANXA6-specific fixation sensitivity or retrieval condition (HPA: tissue IHC profile; UniProt P08133 record). Treat retrieval as an assay setting to verify with the IHC-validated antibody, without attributing a fixation effect to ANXA6 (standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No cytoplasmic staining in an expected positive population. | The assay may have failed, or the selected section may lack the intended cells; HPA reports adrenal glandular cells as High (HPA: tissue IHC). | Verify the cells on the counterstained section, examine a positive control, then review the IHC-validated antibody's retrieval, dilution, and detection settings (standard IHC practice). |
| Only nuclei stain in otherwise intact cells. | The compartment conflicts with the reported cytoplasmic pattern (HPA: tissue IHC profile; UniProt P08133: cytoplasm). | Compare with a known positive section and a control without primary antibody; reassess antibody specificity and detection background before scoring (standard IHC practice). |
| Strong signal appears in a population HPA calls Not detected. | For example, HPA reports esophageal squamous epithelial cells as Not detected; unexpected signal may reflect cross-reactivity or detection activity (HPA: esophagus tissue IHC; standard IHC practice). | Check the cell identity and a control without primary antibody, and compare the staining with an HPA-reported positive population before assigning ANXA6 positivity (standard IHC practice). |
| Chromogen coats large areas or persists between cells. | Diffuse background can conceal cell-associated cytoplasmic staining (HPA: tissue IHC profile; standard IHC practice). | Inspect a control without primary antibody, wash and blocking steps, and any endogenous enzyme blocking required by the chromogenic detection system (standard IHC practice). |
| Different cells in one tissue stain at different intensities. | HPA reports staining by cell population and describes low tissue RNA specificity; a tissue name alone does not set one intensity for every cell (HPA: tissue IHC; HPA: RNA specificity). | Score identifiable cell populations and their cytoplasmic signal separately; compare each with its HPA entry instead of averaging the whole section (HPA: tissue IHC; standard IHC practice). |
| A discrete basal-body focus seen by IF/ICC is absent from chromogenic IHC. | Basal-body localization comes from ICC-IF, whereas HPA describes tissue IHC as ubiquitously cytoplasmic (HPA: subcellular ICC-IF; HPA: tissue IHC profile). | Judge the IHC section by cell-associated cytoplasmic staining in appropriate populations; use the separate IF/ICC guide when assessing that subcellular focus (HPA: tissue IHC; HPA: subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing ANXA6 staining
M03735-1 · IHCIHC analysis of Annexin VI using anti-Annexin VI antibody (M03735-1). Annexin VI was detected in a paraffin-embedded section of human liver
M03735-1 · IHCIHC analysis of Annexin VI using anti-Annexin VI antibody (M03735-1). Annexin VI was detected in a paraffin-embedded section of human lung c
M03735-1 · IFIF analysis of Annexin VI using anti-Annexin VI antibody (M03735-1). Annexin VI was detected in an immunocytochemical section of T-47D cells
A03735 · IHCIHC analysis of Annexin VI/ANXA6 using anti-Annexin VI/ANXA6 antibody (A03735). Annexin VI/ANXA6 was detected in a paraffin-embedded section
A03735 · IHCIHC analysis of Annexin VI/ANXA6 using anti-Annexin VI/ANXA6 antibody (A03735). Annexin VI/ANXA6 was detected in a paraffin-embedded section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197043-ANXA6/tissue/cerebral+cortex#imid_2716020
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197043-ANXA6/tissue/colon#imid_2715946
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197043-ANXA6/tissue/liver#imid_2715947
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000197043-ANXA6/tissue/pancreas#imid_2715898
HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA009977; green = ANXA6, blue = nucleus, red = microtubules.
HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA009977; green = ANXA6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ANXA6 IHC & IF
🧪Run adrenal gland first: glandular cells should show High ANXA6 staining (HPA: adrenal gland, glandular cells, High). Use gallbladder glandular cells as the negative tissue (HPA: gallbladder, glandular cells, Not detected); on the adrenal slide, cells without specific staining should remain at background level, but the supplied HPA row does not identify a particular internal negative cell type (HPA: adrenal gland row).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Esophagus (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ANXA6 in HAP1, Rh30, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a matched mouse IgG isotype control for the catalog antibody (selected-SKU caption: mouse primary antibody); use an ANXA6 knockout specimen, if available, as a biological negative control (standard IHC practice). Check for endogenous peroxidase and biotin background when using the caption’s biotinylated secondary, streptavidin–biotin detection and DAB (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU tissue-IHC caption does not state a fixative (selected-SKU caption: fixative not stated). That caption documents heat-mediated retrieval in EDTA at pH 8.0 before paraffin-section staining; it does not establish that retrieval is required under every condition (selected-SKU caption: EDTA retrieval, paraffin section). ICC-IF images exist for ANXA6, but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC (HPA: subcellular ICC-IF cell lines; selected-SKU caption: paraffin section); assess endogenous peroxidase or biotin background with the caption’s DAB/SABC detection (standard IHC practice; selected-SKU caption: SABC and DAB).
HPA tissue IHC evidence for ANXA6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Pending external verification.). 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 | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Pancreas | Pancreatic endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Small intestine | Endocrine cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ANXA6 IHC Tips
Troubleshoot ANXA6 staining in paraffin sections using the catalog antibody’s tissue IHC conditions and compartment specific controls (datasheet M03735-1; HPA tissue IHC).
Which retrieval condition should I try first for weak ANXA6 staining?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet M03735-1). The catalog antibody produced staining in a paraffin embedded human liver section after that treatment, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet M03735-1). If staining remains weak, compare a second retrieval condition on matched sections while holding antibody concentration, detection chemistry, and development time constant (standard IHC practice). Judge improvement by cytoplasmic signal in an expected cell population and by the absence of a parallel rise in background (HPA tissue IHC; standard IHC practice).
Could fixation explain weak ANXA6 staining in my paraffin sections?
ANXA6 specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin section caption does not report its fixative (datasheet M03735-1). Record the actual fixative and fixation duration for each specimen, then compare sections processed with matched retrieval, antibody exposure, and chromogen development (standard IHC practice). Include a tissue control with an expected positive cell population, such as placental endothelial cells, to detect a run wide loss of signal (HPA tissue IHC; standard IHC practice). Treat any fixation effect as an observation from that controlled comparison, rather than an established property of ANXA6 (standard IHC practice).
Where should convincing ANXA6 staining appear within cells?
Look primarily for cytoplasmic staining in tissue sections: the tissue profile describes ubiquitous cytoplasmic expression, while cellular imaging places ANXA6 mainly in the cytosol (HPA tissue IHC; HPA subcellular). An additional basal body location is reported in cellular imaging, and melanosome fraction identification comes from mass spectrometry (HPA subcellular; UniProt P08133). ANXA6 has no annotated transmembrane segment, so a purely crisp membrane outline deserves independent validation (UniProt P08133 topology; standard IHC practice). Compare the pattern with cell morphology and a no primary control before interpreting punctate, nuclear, or edge limited deposits as specific signal (standard IHC practice).
How can isoforms or epitope masking affect an ANXA6 IHC result?
ANXA6 has 2 annotated isoforms and multiple modified residues, including phosphorylation and acetylation sites (UniProt P08133). The supplied caption specifies the catalog antibody’s staining conditions but gives no epitope map or isoform reactivity, so a negative section cannot establish which isoform is absent (datasheet M03735-1). If an isoform specific conclusion matters, obtain epitope documentation or compare independently characterized antibodies on matched sections (standard IHC practice). Keep retrieval and detection conditions aligned during that comparison, because changes in accessibility or signal amplification can mimic a biological difference (standard IHC practice).
How should I adapt the ANXA6 question to multiplex IF?
Use this entry to plan a separate IF validation: the selected product evidence demonstrates chromogenic staining in a paraffin section, with no IF conditions supplied (datasheet M03735-1). Pair ANXA6 with a validated marker of an expected population, such as placental endothelial cells, and check that the signals occupy the intended cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and favor a far red channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). Because the expected dominant signal is cytosolic and ANXA6 has no transmembrane segment, optimize permeabilisation for intracellular access without assuming an extracellular epitope (HPA subcellular; UniProt P08133 topology; standard IF practice).
What should I check when ANXA6 DAB staining looks diffuse or excessive?
First compare a no primary section with the stained section, and inspect whether deposits follow tissue edges, folds, or damaged areas (standard IHC practice). The catalog example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and biotin based DAB detection (datasheet M03735-1). Check peroxidase blocking as a general chromogenic IHC step, and consider endogenous biotin when troubleshooting a biotin based detection system (standard IHC practice; datasheet M03735-1). Adjust primary concentration or DAB development on matched sections only after checking controls, so reduced background does not conceal expected cytoplasmic staining (HPA tissue IHC; standard IHC practice).
How should I score ANXA6 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because the tissue profile is cytoplasmic and staining varies by cell type (HPA tissue IHC). For cell based comparisons, record the percentage of positive cells and intensity with an H score, using the same thresholds and chromogen development across slides (standard IHC practice). For spatial comparisons, count positive cells per mm² of viable, evaluable tissue and report the sampled area (standard IHC practice). Normalize results to the corresponding cell population or viable tissue area, and exclude folds, necrosis, and section edges using prespecified rules (standard IHC practice).
How can I distinguish genuine ANXA6 positivity from staining artefact?
Prioritize cytoplasmic signal in a plausible cell population: HPA reports high staining in placental endothelial and smooth muscle cells, with mainly cytosolic localization in cellular imaging (HPA tissue IHC; HPA subcellular). Treat isolated nuclear deposits or a uniform membrane outline cautiously, since the supplied localization evidence centers on the cytosol and ANXA6 lacks a transmembrane segment (HPA subcellular; UniProt P08133 topology). Compare questionable regions with section edges and necrotic areas, then inspect a no primary control for endogenous enzyme or detection background (standard IHC practice). A reproducible compartment pattern across intact cells and matched control sections provides stronger support than intensity alone (standard IHC practice).
Boster reagents
Best ANXA6 / Annexin A6 IHC Antibodies
The catalog shows paraffin-section IHC images from human, mouse and rat tissues, plus IF/ICC imaging in human T-47D cells (A03735 and M03735-1 image captions).
Real IHC data
Anti-Annexin VI Antibody ® (monoclonal, 4C4)
Cat # M03735-1
Real IHC data
Anti-Annexin VI/ANXA6 Antibody ®
Cat # A03735
A03735 has IHC images from human lung and thyroid cancers, mouse lymphade and rat colon (A03735 IHC image captions). M03735-1 has IHC images from human liver, lung cancer and renal clear cell carcinoma and mouse pancreas, plus IF/ICC imaging in T-47D cells (M03735-1 image captions).
Which to pick: For tissue IHC across species, choose A03735: its own paraffin-section captions cover human, mouse and rat samples (A03735 IHC image captions). For IF/ICC, choose monoclonal M03735-1, whose application list includes both and whose IF caption documents T-47D cells (M03735-1 catalog applications; M03735-1 IF image caption). M03735-1 also has paraffin-section IHC images from human and mouse samples; neither SKU’s IHC captions report a fixative (M03735-1 IHC image captions; A03735 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P08133 (ANXA6_HUMAN, Annexin A6).
- Human Protein Atlas. ANXA6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ANXA6 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the basal body..
- Human Protein Atlas. ANXA6 antibody validation summary (4 antibodies).
- EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence. Oxidative medicine and cellular longevity 2022 — PMC9492406.
- SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1-involved EMT in hepatocellular carcinoma. Cell communication and signaling : CCS 2024 — PMC10986105.
- Breast Cancer Stem Cell-Derived ANXA6-Containing Exosomes Sustain Paclitaxel Resistance and Cancer Aggressiveness in Breast Cancer. Frontiers in cell and developmental biology 2021 — PMC8523856.
- Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer. Cell death & disease 2021 — PMC8266800.
- PubMed PMID:3258820 — UniProt-cited evidence.
- PubMed PMID:2963335 — UniProt-cited evidence.
- PubMed PMID:2528541 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ANXA6 (Annexin A6). Expected localisation Cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03735-1). Validated IHC antibody: M03735-1 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Esophagus. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver tissue; fixative not specified (datasheet M03735-1); 2. Fixation: Image fixative and duration unreported (datasheet M03735-1); 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 M03735-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03735-1); 8. Primary antibody: Mouse monoclonal (clone 4C4) anti-ANXA6, 2 μg/ml (datasheet M03735-1); 9. Primary incubation: Overnight at 4 °C (datasheet M03735-1). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Some cell populations have no detectable signal (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unspecified (UniProt); Regulation: Low tissue specificity; no regulator annotated (HPA tissue IHC; UniProt). Evidence: UniProt P08133, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the basal…, Datasheet M03735-1, PMC9492406, PMC8266800. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ANXA6/immunohistochemistry-ihc-guide.