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CA9 / Carbonic anhydrase 9 · IHC design guide
Design Immunohistochemistry for CA9
Plan CA9 chromogenic IHC in paraffin sections using the IHC-validated antibody at 2 μg/ml (datasheet A01083-3). Assess glandular membrane staining in stomach or gallbladder (HPA tissue IHC), with a negative control for interpretation (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CA9 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 | Membranous in gastrointestinal glandular cells (HPA tissue IHC) | |
| Staining pattern | Glandular cell membrane staining in GI mucosa and gallbladder (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01083-3) | |
| Positive control | Gallbladder+3 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Normal stomach and gallbladder glands can stain strongly (HPA tissue IHC) | |
| Regulation | Expression regulation is not specified (UniProt) | |
| Isoform / epitope | No listed isoforms; extracellular versus cytoplasmic epitope matters (UniProt) |
Section 1
Recommended CA9 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A01083-3) is accompanied by four published CA9 IHC protocols (PMC6047828; PMC3875845; PMC8168507; PMC8804613).
| Sample | Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet A01083-3) |
| Fixation | Image fixative and duration unreported (datasheet A01083-3); 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 A01083-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01083-3) |
| Primary antibody | Rabbit anti-CA9, 2-5 μg/ml (datasheet A01083-3) |
| Primary incubation | Overnight at 4 °C (datasheet A01083-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01083-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CA9-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Highly selective membranous expression in the gastrointestinal mucosa and gall bladder. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A01083-3); follow each article’s stated retrieval when reproducing its protocol (PMC6047828; PMC3875845; PMC8168507).
Section 2
What Is the Expected CA9 Staining Pattern?
CA9 is a membrane protein with an extracellular region spanning residues 38–414 and a transmembrane segment at 415–435 (UniProt Q16790 topology). In tissue IHC, expect selective membranous staining of gastrointestinal and gallbladder glandular cells, strongest in stomach and gallbladder (HPA: Enhanced reliability; High in stomach and gallbladder glandular cells). UniProt also reports nuclear and nucleolar localization, which warrants separate assessment if seen (UniProt Q16790: subcellular location).
| Distinct membrane outlines in glandular cells, strong in stomach or gallbladder and moderate in duodenum or small intestine (HPA: tissue IHC). | This matches the reported tissue pattern (HPA: High in stomach and gallbladder; Medium in duodenum and small intestine). Score the glandular cell membranes rather than assigning a whole-section intensity, since the expected result is cell selective (HPA: highly selective membranous expression). |
| Predominantly nuclear or nucleolar chromogen with little glandular membrane staining (HPA: tissue IHC; UniProt Q16790: subcellular location). | The slide does not reproduce HPA’s tissue IHC pattern (HPA: membranous expression). UniProt reports nuclear and nucleolar CA9, so nuclear staining alone is not proof of artefact; check controls and antibody validation before treating it as a tissue IHC positive (UniProt Q16790: nucleus and nucleolus; standard IHC practice). |
| Staining appears mainly in adipocytes, hematopoietic cells, or respiratory epithelial cells (HPA: Not detected in the listed cell types). | These cells are poor CA9-positive references in the supplied tissue survey (HPA: Not detected). Consider antibody cross-reactivity or endogenous chromogenic detection activity; compare a known-positive glandular section with negative and detection-only controls (standard IHC practice). |
| A diffuse haze covers glands, stroma, and empty areas instead of outlining glandular cell membranes (HPA: expected membranous pattern). | A broad background pattern cannot establish CA9 localization (HPA: highly selective membranous expression). Check nonspecific antibody binding, detection reagent background, and washing with an appropriate negative control before scoring the section (standard IHC practice). |
| No membrane signal is visible in stomach or gallbladder glandular cells (HPA: High in both cell populations). | The run has failed its expected positive-tissue check, although a single negative section cannot identify the cause (HPA: High in stomach and gallbladder glandular cells; standard IHC practice). Review tissue identity, antibody validation, retrieval, and detection controls before interpreting other negative sections (standard IHC practice). |
💡Expected CA9 appearanceCall a positive result when glandular cell membranes stain strongly in stomach or gallbladder, or moderately in duodenum or small intestine; diffuse haze or staining chiefly in HPA-negative cell types does not match that result (HPA: tissue IHC levels and membranous profile).
| Membrane topology and compartment (UniProt Q16790 topology; HPA: tissue IHC). | CA9 has a large extracellular region, residues 38–414, followed by one transmembrane segment, residues 415–435 (UniProt Q16790 topology). The reported tissue IHC pattern is membranous (HPA: tissue IHC); topology alone does not specify a particular antibody’s epitope or retrieval requirement. |
| Choice of positive and negative tissue references (HPA: tissue IHC). | Stomach and gallbladder glandular cells are High; duodenum and small intestine glandular cells are Medium (HPA: tissue IHC). Adipocytes in adipose tissue and hematopoietic cells in bone marrow are Not detected (HPA: tissue IHC). Match the reference to the cell type being scored. |
| Antibody-specific IHC evidence (HPA: antibody validation). | HPA lists IHC as Enhanced for HPA055207, CAB005100, and CAB017107 (HPA: antibody validation). That supports comparison with the reported tissue pattern; it does not establish identical behavior for an unlisted catalog antibody or a particular antigen-retrieval condition. |
| IF/ICC Q: What localization should I expect? (HPA: subcellular ICC-IF). | A: HPA reports supported plasma-membrane localization in ICC-IF and lists images from A-431 and U2OS (HPA: subcellular ICC-IF). This is a localization reference, not an IF/ICC protocol or evidence that every cell will stain. |
| Situation | Likely cause | Next action |
|---|---|---|
| Stomach or gallbladder positive-control glands lack membrane staining (HPA: High in both). | The tissue, primary-antibody step, retrieval, or chromogenic detection may have failed; the slide alone cannot distinguish them (standard IHC practice). | Confirm glandular tissue identity and review the antibody’s IHC validation, retrieval conditions, and detection controls; repeat with a documented positive section (HPA: tissue IHC; standard IHC practice). |
| Only nuclei or nucleoli stain in a tissue section (UniProt Q16790: nuclear localization). | This differs from HPA’s membranous tissue pattern, although UniProt also reports nuclear and nucleolar CA9 (HPA: tissue IHC; UniProt Q16790: subcellular location). | Record the compartment separately, inspect positive and negative controls, and avoid scoring nuclear-only staining as the established tissue IHC pattern (HPA: membranous profile; standard IHC practice). |
| HPA-negative cell types appear positive, such as adipocytes in adipose tissue (HPA: Not detected). | Cross-reactivity or endogenous chromogenic activity is possible; HPA’s negative observation does not diagnose which cause applies (HPA: tissue IHC; standard IHC practice). | Compare with a detection-only control and a glandular positive control; review blocking and the chromogenic detection system if the unexpected signal persists (standard IHC practice). |
| Diffuse chromogen obscures cell borders (HPA: expected selective membranous profile). | Nonspecific binding, detection background, or inadequate washing can obscure a localized signal (standard IHC practice). | Inspect negative controls, blocking, primary-antibody concentration, and wash steps before calling any glandular cells positive (standard IHC practice). |
| Duodenal or small-intestinal glands look weaker than stomach glands (HPA: Medium versus High). | That relative difference is consistent with the reported tissue levels and does not by itself indicate a failed run (HPA: tissue IHC). | Judge each section against its own expected cell type and compartment; use stomach or gallbladder glands for a stronger positive reference (HPA: tissue IHC). |
| A proposed antigen-retrieval change is being justified as CA9-specific (UniProt Q16790: topology; HPA: tissue IHC). | The supplied sources report localization and tissue staining, but no target-specific fixation sensitivity or retrieval comparison (UniProt Q16790; HPA: tissue IHC). | Treat retrieval changes as general IHC optimization and compare controlled sections; do not infer a CA9-specific fixation effect from topology or HPA intensity (standard IHC practice; UniProt Q16790 topology; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing CA9 staining
A01083-3 · IHCIHC analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3). Carbonic Anhydrase 9/CA9 was detected in a
A01083-3 · IHCIHC analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3). Carbonic Anhydrase 9/CA9 was detected in a
M01083-5 · IHCHuman tonsil was stained with Anti-Carbonic Anhydrase 9 rabbit antibody
M01083-5 · IHCRat stomach was stained with Anti-Carbonic Anhydrase 9 rabbit antibody
M01083-1 · IHCImmunohistochemical analysis of paraffin-embedded human kindey cancer, using CA9 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000107159-CA9/tissue/cerebral+cortex#imid_17269135
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000107159-CA9/tissue/stomach+1#imid_17269035
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000107159-CA9/tissue/colon#imid_17269061
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000107159-CA9/tissue/liver#imid_17269062
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB017107; green = CA9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB017107; green = CA9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CA9 IHC & IF
🧪Run stomach first: its glandular cells should stain (HPA: High in stomach glandular cells). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected in adrenal gland glandular cells); on the stomach slide, assess adjacent non-glandular cells for absent or much weaker signal than the glandular cells (HPA: High in stomach glandular cells).
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CA9 in A-431, U2OS, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality and concentration where known (selected-SKU caption: rabbit anti-CA9); use a CA9-knockout specimen as a biological negative control. Quench endogenous peroxidase for the stomach section before chromogenic detection (selected-SKU caption: HRP/DAB; HPA: High in stomach glandular cells).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). Start with paraffin-section IHC using heat retrieval in EDTA at pH 8.0; the caption documents this workflow but does not establish whether retrieval is required, and the supplied evidence does not show that frozen sections or IF are easier (selected-SKU caption: paraffin section, heat retrieval in EDTA pH 8.0). In stomach sections, distinguish glandular-cell staining from luminal material or background when scoring (HPA: High in stomach glandular cells).
HPA tissue IHC evidence for CA9
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 |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Small intestine | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CA9 IHC Tips
Troubleshoot CA9 staining in paraffin sections by checking retrieval, controls, membrane localisation and how positive cells are scored.
What retrieval should I try first if CA9 staining is weak?
Use heat mediated retrieval in EDTA at pH 8.0 first (datasheet A01083-3). The selected paraffin section example used that retrieval before 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A01083-3). If staining remains weak, adjust heating duration on matched sections while keeping the buffer and detection steps constant, and watch for tissue damage (standard IHC practice). Include a known positive glandular section, since stomach and gallbladder glandular cells show high CA9 staining (HPA tissue IHC). A weak result without a working positive control cannot distinguish retrieval failure from low expression (standard IHC practice).
Could fixation explain weak CA9 staining in my paraffin sections?
The selected CA9 image documents a paraffin embedded section but does not state its fixative, so target specific fixation sensitivity is unknown (datasheet A01083-3). Record the fixative and fixation time for each specimen, then compare sections processed with the same retrieval and antibody conditions (standard IHC practice). For a controlled comparison, start with EDTA at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C, as used in the selected image (datasheet A01083-3). Check morphology alongside staining: damaged or detached epithelium can make a negative region difficult to judge (standard IHC practice). Use a positive section in the same run before assigning weak signal to fixation (standard IHC practice).
Which staining compartments should count as CA9 positive?
Prioritise a crisp cell surface pattern in glandular epithelium: the tissue profile is highly selective and membranous in gastrointestinal mucosa and gallbladder (HPA tissue IHC). CA9 spans the membrane at residues 415–435, with residues 38–414 outside the cell and 436–459 inside (UniProt Q16790 topology). UniProt also reports microvillus surface and nuclear, particularly nucleolar, localisation, while the supported subcellular location is plasma membrane (UniProt Q16790 subcellular; HPA subcellular). Score membrane and nuclear patterns separately rather than pooling them into one positive category (standard IHC practice). Diffuse staining that obscures cell boundaries warrants review of background controls and tissue morphology (standard IHC practice).
How could epitope location change the CA9 staining pattern?
Check the antibody's mapped epitope, if available, before interpreting an unexpected compartment or changing tissue processing (standard IHC practice). CA9 has no listed isoforms; its extracellular region spans residues 38–414, and its cytoplasmic tail spans 436–459 (UniProt Q16790 isoforms and topology). The protein has glycosylation sites at residues 115 and 346, plus a reported phosphotyrosine at 449 (UniProt Q16790 modified residues). These features identify regions to consider during epitope review; they do not establish which region the catalog antibody recognises (UniProt Q16790 topology; datasheet A01083-3). Compare staining after the documented EDTA pH 8.0 retrieval with appropriate positive and negative sections (datasheet A01083-3; standard IHC practice).
How should I check CA9 localisation in a multiplex IF experiment?
Plan a separate IF validation with a marker that identifies the expected glandular cells; stomach and gallbladder glandular cells show high CA9 staining by tissue IHC (HPA tissue IHC). Choose fluorophores after examining the specimen's autofluorescence, and include single stain controls to assess channel bleed through (standard IF practice). For an antibody recognising residues 38–414 outside the cell, test whether permeabilisation is needed; an epitope within residues 436–459 requires membrane access (UniProt Q16790 topology; standard IF practice). The catalog antibody's epitope side is unspecified in the supplied evidence, so do not assume either condition will work (datasheet A01083-3). Compare any IF pattern with the supported plasma membrane location (HPA subcellular).
What should I change when CA9 DAB staining looks diffuse?
Inspect a section without primary antibody to identify signal from detection reagents or endogenous peroxidase, and apply a peroxidase block before DAB development (standard IHC practice). The selected example used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and an anti rabbit secondary for 30 minutes at 37°C (datasheet A01083-3). If the no primary control is clean, adjust primary concentration or incubation and compare matched sections using the same DAB development time (standard IHC practice). Examine tissue folds, edges and necrotic areas separately from intact epithelium (standard IHC practice). Genuine tissue staining should be assessed against the predominantly membranous glandular pattern (HPA tissue IHC).
How should I score CA9 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and cell population before scoring, then record the percentage of positive glandular cells and membrane intensity on the same criteria across sections (standard IHC practice). An H score can combine percentages at intensity grades 0–3, giving a range of 0–300 (standard IHC practice). Normalise positive counts to the number of evaluable cells, or report positive cell density per mm² of evaluable tissue when area is the intended denominator (standard IHC practice). Exclude folds, necrosis and missing epithelium consistently, and keep nuclear staining separate from membrane staining (standard IHC practice; HPA subcellular). Document the scoring threshold using controls from the same staining run (standard IHC practice).
How can I tell true CA9 staining from artefact?
Look for staining that follows intact cell boundaries in the expected cells: HPA reports highly selective membranous expression in gastrointestinal mucosa and gallbladder (HPA tissue IHC). Stomach and gallbladder glandular cells provide high staining comparators, whereas adipocytes in adipose tissue are reported as not detected (HPA tissue IHC). Review isolated edge signal, necrotic areas and positive no primary controls as possible processing or endogenous enzyme artefacts before calling cells positive (standard IHC practice). Nuclear or nucleolar staining deserves separate assessment because UniProt reports those locations, while HPA supports plasma membrane localisation (UniProt Q16790 subcellular; HPA subcellular). Confirm uncertain regions against morphology and a matched positive control (standard IHC practice).
Boster reagents
Best CA9 / Carbonic anhydrase 9 IHC Antibodies
Three anti-CA9 antibodies have IHC figures from human tissues, with mouse and rat tissue also represented (image captions: A01083-3, M01083-1, M01083-5); M01083-5 lists IF validation (catalog: M01083-5).
Real IHC data
Anti-Carbonic Anhydrase 9/CA9 Antibody ®
Cat # A01083-3
Real IHC data
Anti-Carbonic Anhydrase 9 Rabbit Monoclonal Antibody
Cat # M01083-5
Real IHC data
Anti-CA9/Ca Ix Rabbit Monoclonal Antibody
Cat # M01083-1
A01083-3 shows paraffin-embedded human colonic and renal adenocarcinoma and mouse stomach; M01083-1 shows paraffin-embedded human kidney cancer (image captions: A01083-3, M01083-1). M01083-5 shows IHC staining of human tonsil, colon carcinoma and stomach, and rat stomach; it also lists IF among its applications (M01083-5 image captions; catalog).
Which to pick: For paraffin-section chromogenic IHC, start with A01083-3: its captions document EDTA retrieval at pH 8.0, 2 μg/ml primary antibody and HRP/DAB detection; the fixative is unreported (A01083-3 image captions). For IF/ICC, M01083-5 is the catalog option because it lists IF at 1:200–1:1000, though no IF figure is supplied (M01083-5 catalog). For work across species, M01083-5 lists human, mouse and rat reactivity, with IHC images from human and rat tissue; its captions do not report processing or fixative (M01083-5 catalog; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16790 (CAH9_HUMAN, Carbonic anhydrase 9).
- Human Protein Atlas. CA9 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CA9 subcellular location (ICC-IF): Localized to the plasma membrane..
- Human Protein Atlas. CA9 antibody validation summary (3 antibodies).
- Circulating hypoxia marker carbonic anhydrase IX (CA9) in patients with hepatocellular carcinoma and patients with cirrhosis. PloS one 2018 — PMC6047828.
- Expression of carbonic anhydrase IX in human fetal joints, ligaments and tendons: a potential marker of mechanical stress in fetal development?. Anatomy & cell biology 2013 — PMC3875845.
- Molecular profiling reveals a hypoxia signature in breast implant-associated anaplastic large cell lymphoma. Haematologica 2021 — PMC8168507.
- Identification of ferroptosis-related genes as potential biomarkers of tongue squamous cell carcinoma using an integrated bioinformatics approach. FEBS open bio 2022 — PMC8804613.
- PubMed PMID:8084592 — UniProt-cited evidence.
- PubMed PMID:10709109 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CA9 (Carbonic anhydrase 9). Expected localisation Membranous in gastrointestinal glandular cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01083-3). Validated IHC antibody: A01083-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet A01083-3); 2. Fixation: Image fixative and duration unreported (datasheet A01083-3); 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 A01083-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01083-3); 8. Primary antibody: Rabbit anti-CA9, 2-5 μg/ml (datasheet A01083-3); 9. Primary incubation: Overnight at 4 °C (datasheet A01083-3). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Normal stomach and gallbladder glands can stain strongly (HPA tissue IHC); Isoform / epitope: No listed isoforms; extracellular versus cytoplasmic epitope matters (UniProt); Regulation: Expression regulation is not specified (UniProt). Evidence: UniProt Q16790, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane., Datasheet A01083-3, PMC6047828, PMC3875845. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CA9/immunohistochemistry-ihc-guide.