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CARD9 / Caspase recruitment domain-containing protein 9 · IHC design guide
Design Immunohistochemistry for CARD9
Expect cytoplasmic CARD9 staining in immune cells (HPA tissue IHC). For paraffin IHC, use the catalog antibody at 0.5–1 μg/ml (datasheet A01410-1) and compare lung macrophages with unstained adipocytes (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CARD9 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 in several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in immune cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01410-1) | |
| Positive control | Testis+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 sections (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining was disregarded (HPA tissue IHC) | |
| Regulation | Expression restricted to phagocyte populations (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage needs sequence review (UniProt) |
Section 1
Recommended CARD9 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A01410-1) with published CARD9 protocols for paraffin sections (PMC4085533), mouse heart (PMC3139445), and human lung adenocarcinoma (PMC7498929).
| Sample | Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A01410-1) |
| Fixation | Image fixative and duration unreported (datasheet A01410-1); 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 A01410-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01410-1) |
| Primary antibody | Rabbit anti-CARD9, 0.5-1μg/ml (datasheet A01410-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01410-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01410-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CARD9-positive staining in elongated or late spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, including immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A01410-1); PMC7498929 reports a Tris-EDTA-citrate pH 7.8 alternative.
Section 2
What Is the Expected CARD9 Staining Pattern?
CARD9 should appear in the cytoplasm of selected immune cells, especially macrophages, monocytes and dendritic cells (UniProt Q9H257: cytoplasm; phagocyte expression). It has no transmembrane segment (UniProt Q9H257: topology). HPA reports cytoplasmic staining in several tissues and rates tissue IHC reliability Enhanced, while noting medium agreement with RNA data and presumed off-target staining that was disregarded (HPA: tissue IHC reliability and profile).
| Cytoplasmic staining in lung macrophages, spleen red-pulp cells or tonsil non-germinal-center cells. | This fits the reported IHC pattern: HPA scores each population Medium (HPA: lung, spleen and tonsil tissue IHC). Judge the stained cells within each section rather than expecting every cell to stain; UniProt describes expression in selected phagocyte populations (UniProt Q9H257: tissue specificity). |
| Predominantly nuclear or sharply membrane-bound staining in cells identified as CARD9-positive. | That compartment conflicts with the annotated cytoplasmic location and lack of a transmembrane segment (UniProt Q9H257: subcellular location and topology). Treat it as a possible localisation artefact and check the same run against a known-positive tissue and its controls (general IHC practice). |
| Strong staining across an unexpected cell population, including cells listed as not detected. | Consider cross-reactivity or endogenous detection activity before assigning those cells CARD9 expression (general IHC practice). HPA lists adipocytes and bronchial basal cells as Not detected, but also reports High staining in testis elongated or late spermatids; cell identity and tissue context matter (HPA: tissue IHC). |
| Diffuse colour over cells and tissue spaces without a clear cytoplasmic cell pattern. | This is difficult to score as CARD9 because the expected location is cytoplasmic and HPA describes expression in selected cells (UniProt Q9H257: subcellular location; HPA: tissue IHC profile). Compare negative and detection-only controls, then review blocking, washing and chromogen development (general IHC practice). |
| No detectable signal in a lung macrophage-rich area or spleen red pulp. | A negative result in a reported Medium-staining population warrants a run-level check before a biological conclusion (HPA: lung and spleen tissue IHC). Confirm that the expected cells are present, then review retrieval, primary-antibody incubation and detection controls (general IHC practice). |
💡Expected CARD9 appearanceCall the result consistent with CARD9 when selected cells show cytoplasmic staining, including Medium staining in lung macrophages; widespread nuclear, membrane-bound or cell-free colour is suspect (UniProt Q9H257: cytoplasm and phagocytes; HPA: lung macrophages, Medium; general IHC practice).
| Cell population and tissue context | HPA reports Medium staining in lung macrophages, spleen red-pulp cells and tonsil non-germinal-center cells, but High staining in testis elongated or late spermatids (HPA: tissue IHC). Use the named cell population when comparing sections; a whole-tissue positive label does not describe every cell (general IHC practice). |
| Compartment and topology | CARD9 is annotated as cytoplasmic, with no transmembrane segment or signal peptide (UniProt Q9H257: subcellular location, topology and processing). These annotations support a cytoplasmic interpretation but do not specify whether chromogenic staining should be smooth or punctate. |
| Antibody evidence and its limit | HPA lists antibody HPA059502 as IHC Enhanced and describes tissue staining as cytoplasmic, with medium agreement between staining and RNA expression; presumed off-target binding was observed and disregarded (HPA: antibody status, reliability and tissue profile). Assess unexpected staining with controls rather than treating Enhanced as proof of specificity (general IHC practice). |
| Isoforms and retrieval | UniProt lists 3 CARD9 isoforms but supplies no antibody epitope or isoform coverage here (UniProt Q9H257: isoforms; HPA: supplied antibody record). The supplied sources also give no CARD9-specific retrieval or fixation-sensitivity result. If signal is absent, optimise retrieval as a general IHC workflow variable, without assigning a CARD9-specific effect (general IHC practice). |
| IF/ICC: should it show the same pattern? | Cytoplasmic localisation is a reasonable expectation from UniProt, but the supplied HPA subcellular record has no main-location assignment or ICC-IF images, and the antibody record gives no ICC validation (UniProt Q9H257: cytoplasm; HPA: subcellular and antibody records). This evidence does not establish an IF/ICC staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected immune-cell staining is absent in lung or spleen. | The positive cells may be missing from the field, or an IHC workflow step may have failed; HPA reports Medium staining in lung macrophages and spleen red-pulp cells (HPA: tissue IHC; general IHC practice). | Find the named cells on a counterstained section; then check a known-positive section, retrieval, primary incubation and detection controls in the same run (general IHC practice). |
| Nuclear staining dominates the putative positive cells. | This disagrees with CARD9's annotated cytoplasmic location (UniProt Q9H257: subcellular location). The supplied sources do not establish nuclear staining as the expected IHC pattern. | Compare the nuclear signal with negative controls and with cytoplasmic staining in a reported positive population before scoring it as CARD9 (HPA: tissue IHC; general IHC practice). |
| Colour appears along cell borders or outside cells. | A membrane-bound pattern conflicts with the reported cytoplasmic location and absence of a transmembrane segment; extracellular colour can also reflect nonspecific detection (UniProt Q9H257: location and topology; general IHC practice). | Review detection-only controls, washing and chromogen development; score only interpretable cell-associated staining in the expected compartment (general IHC practice). |
| Many cells stain, including an HPA Not detected population. | Cross-reactivity or endogenous detection activity is possible; HPA reports some tissue cell populations as Not detected and notes presumed off-target staining in its reliability summary (HPA: tissue IHC and reliability; general IHC practice). | Verify cell identity and compare negative and detection-only controls. Interpret a result in the context of the specific HPA cell population rather than a tissue-wide label (HPA: tissue IHC; general IHC practice). |
| The section has uniform haze that obscures cell boundaries. | Background from the staining workflow can obscure the cell-specific cytoplasmic pattern described for CARD9 (HPA: tissue IHC profile; general IHC practice). | Check background in control sections and review blocking, washes and chromogen development before changing the biological interpretation (general IHC practice). |
| Testis spermatids stain strongly, despite the phagocyte-focused expectation. | This is an observed HPA result: elongated or late spermatids are scored High, while UniProt describes expression as restricted to several phagocyte populations (HPA: testis tissue IHC; UniProt Q9H257: tissue specificity). | Record the cell identity and compartment, and assess controls. Do not discard the signal solely because it falls outside the phagocyte-focused expectation; HPA also notes presumed off-target staining in its reliability assessment (HPA: tissue IHC and reliability; general IHC practice). |
Experiment evidence
IHC & IF images showing CARD9 staining
A01410-1 · IHCIHC analysis of CARD9 using anti-CARD9 antibody (A01410-1). CARD9 was detected in paraffin-embedded section of human rectal cancer tissue. H
A01410-1 · IFIF analysis of CARD9 using anti-CARD9 antibody (A01410-1). CARD9 was detected in immunocytochemical section of A549 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000187796-CARD9/tissue/cerebral+cortex#imid_16427211
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000187796-CARD9/tissue/colon#imid_16427145
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000187796-CARD9/tissue/liver#imid_16427154
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000187796-CARD9/tissue/kidney#imid_16427147
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CARD9 IHC & IF
🧪Run lung first and look for staining in its macrophages (HPA: Medium in lung macrophages; UniProt Q9H257: expression in phagocytes). Use adrenal gland glandular cells as the negative tissue compartment (HPA: Not detected in adrenal gland glandular cells); on the lung slide, compare macrophages with neighboring nonmacrophage cells, which should remain near background (UniProt Q9H257: expression restricted to phagocyte populations).
Positive control tissue: Testis (Elongated or late spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CARD9; derive a cell-line control from the positive tissue's cell type (Elongated or late spermatids) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody); use CARD9-knockout material as a biological negative when available. For the caption’s biotin-based DAB method, block endogenous peroxidase and check endogenous biotin (selected-SKU caption: streptavidin–biotin complex with DAB); for IF, assess tissue autofluorescence with a no-primary control (standard IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in EDTA, pH 8.0, but does not establish that CARD9 staining depends on this retrieval method (selected-SKU caption: EDTA retrieval). Matched frozen-section and IF/ICC results are absent, so neither can be judged easier; in lung IHC, distinguish brown macrophage pigment from DAB signal using the negative controls (HPA subcellular: no ICC-IF images; standard IHC practice).
HPA tissue IHC evidence for CARD9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center 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 → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CARD9 IHC Tips
Use the catalog antibody’s paraffin section IHC conditions as a starting point, then judge staining by compartment, cell type, and controls.
Which retrieval condition should I start with for CARD9 paraffin section IHC?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet A01410-1). The selected paraffin section example used that condition before incubation with 1 μg/mL catalog antibody overnight at 4°C (A01410-1 caption). Keep section thickness, heating, cooling, and detection conditions consistent while comparing retrieval runs, because each can change apparent staining intensity (standard IHC practice). If staining remains weak despite a working positive control, compare a second retrieval condition as a fallback and assess both signal and tissue preservation (standard IHC practice). Judge improvement by cytoplasmic staining in expected immune cells rather than stronger diffuse color alone (UniProt Q9H257 subcellular; HPA tissue IHC).
How should I troubleshoot CARD9 staining when fixation varies between paraffin blocks?
Target specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin section caption does not state its fixative (A01410-1 caption). Record the fixative and fixation duration for each block, and compare blocks with similar processing before attributing intensity differences to CARD9 abundance (standard IHC practice). Run a reference section alongside each batch using the documented EDTA pH 8.0 retrieval and 1 μg/mL primary condition (datasheet A01410-1; A01410-1 caption). Examine morphology and cytoplasmic staining together, since damaged tissue can make a visually strong chromogenic result difficult to interpret (standard IHC practice; UniProt Q9H257 subcellular). Do not infer a fixation preference from reported tissue staining patterns (HPA tissue IHC).
What staining pattern is plausible for CARD9 in paraffin sections?
Expect predominantly cytoplasmic signal because CARD9 is annotated in the cytoplasm and has no transmembrane segment (UniProt Q9H257 subcellular and topology). Evaluate staining within individual cells rather than treating color across the whole section as equivalent evidence of expression (standard IHC practice). Macrophages and other phagocyte populations are biologically plausible positive cells, and lung macrophages show medium staining in the tissue atlas (UniProt Q9H257 tissue specificity; HPA tissue IHC). Use morphology and, where needed, an adjacent immune cell marker to distinguish those cells from neighboring tissue elements (standard IHC practice). Predominantly nuclear, crisp membrane, or acellular staining warrants a specificity check before scoring it as CARD9 (UniProt Q9H257 subcellular; standard IHC practice).
Could CARD9 isoforms or epitope accessibility explain inconsistent IHC staining?
CARD9 has 3 annotated isoforms, but the supplied antibody caption does not identify its recognized epitope or establish equal detection of those isoforms (UniProt Q9H257 isoforms; A01410-1 caption). Its CARD domain spans residues 6–98, and the protein has annotated modified residues, including phosphorylation sites (UniProt Q9H257 domains and modified residues). Those annotations alone do not establish which feature changes staining by this antibody (UniProt Q9H257; A01410-1 caption). If sections differ unexpectedly, compare retrieval and antibody conditions on matched controls before proposing an isoform explanation (standard IHC practice). Ask for epitope mapping or independent validation if an isoform specific interpretation is essential (standard IHC practice).
How can IF help assess an ambiguous CARD9 chromogenic IHC pattern?
Use IF as a complementary localisation check, pairing CARD9 with a validated marker for the expected phagocyte population (UniProt Q9H257 tissue specificity; standard IF practice). Choose fluorophores after checking tissue autofluorescence and reserve a spectrally distinct channel for the cell marker (standard IF practice). Because CARD9 is cytoplasmic and lacks a transmembrane segment, permeabilise cells sufficiently for intracellular antibody access while preserving morphology (UniProt Q9H257 subcellular and topology; standard IF practice). Include single stain and secondary only controls to assess bleed through and nonspecific fluorescence (standard IF practice). The paraffin section IHC caption supplies no IF fixation, permeabilisation, or antibody dilution condition, so validate those settings separately (A01410-1 caption).
How do I reduce diffuse brown background without losing CARD9 signal?
First compare a no primary control with the stained section to identify color arising from the detection system or tissue itself (standard IHC practice). The selected example used 10% goat serum block, a biotinylated secondary, a streptavidin biotin complex, and DAB (A01410-1 caption). Check endogenous peroxidase and endogenous biotin background when using that detection scheme, then optimize the relevant blocking step and washes (standard IHC practice). If background tracks primary antibody concentration, titrate around the documented 1 μg/mL condition while retaining a positive control (A01410-1 caption; standard IHC practice). Preserve discernible cytoplasmic cell staining when selecting the final condition (UniProt Q9H257 subcellular; standard IHC practice).
What is a defensible way to quantify CARD9 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment as cytoplasm and identify the cell population before measuring staining (UniProt Q9H257 subcellular and tissue specificity). For a defined population, report the percentage of positive cells and an H-score based on intensity categories; for spatial comparisons, report positive cell density per mm² (standard IHC practice). Normalize counts to the sampled viable tissue area and, where cell composition varies, to the number of eligible cells in that compartment (standard IHC practice). Keep retrieval, detection, exposure to DAB, and scoring thresholds consistent across compared sections (standard IHC practice). Report cell composition alongside intensity, because CARD9 expression is concentrated in phagocyte populations (UniProt Q9H257 tissue specificity).
How can I distinguish genuine CARD9 staining from artefact?
A credible result places staining chiefly in cell cytoplasm and in plausible phagocyte populations (UniProt Q9H257 subcellular and tissue specificity). Check whether the same pattern appears away from section edges and necrotic areas, where nonspecific color can distort interpretation (standard IHC practice). A no primary control helps identify endogenous enzyme or detection background, particularly when reading a DAB section (standard IHC practice). Treat dominant nuclear, sharply membranous, or acellular deposits as suspect until supported by an independent antibody or matched control (UniProt Q9H257 subcellular and topology; standard IHC practice). The tissue atlas reports presumed off target binding, so its staining pattern alone cannot establish specificity (HPA tissue IHC reliability description).
Boster reagents
Best CARD9 / Caspase recruitment domain-containing protein 9 IHC Antibodies
The catalog antibody has IHC data from human rectal cancer paraffin sections and IF data from A549 cells (A01410-1 image captions); catalog reactivity is Human (catalog: A01410-1 reactivity).
Real IHC data
Anti-CARD9 ® Antibody
Cat # A01410-1
A01410-1 has IHC data from a paraffin section of human rectal cancer tissue (A01410-1 IHC caption). The same SKU has IF data from A549 cells (A01410-1 IF caption).
Which to pick: Choose A01410-1 for paraffin-section IHC; its IHC caption documents heat retrieval in EDTA at pH 8.0 and primary antibody at 1 μg/ml (A01410-1 IHC caption). The same SKU supports IF/ICC selection through its application list and A549 IF image (catalog: A01410-1 applications; A01410-1 IF caption). No listed SKU has catalog reactivity beyond Human, and the IHC caption does not report the fixative (catalog: A01410-1 reactivity; A01410-1 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H257 (CARD9_HUMAN, Caspase recruitment domain-containing protein 9).
- Human Protein Atlas. CARD9 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CARD9 subcellular location (ICC-IF): Highest expression in THP-1: 63.3 nTPM.
- Human Protein Atlas. CARD9 antibody validation summary (1 antibodies).
- Caspase recruitment domain family member 9 expression is a promising biomarker in esophageal squamous cell carcinoma. Annals of gastroenterological surgery 2020 — PMC7105841.
- Tumor cell-activated CARD9 signaling contributes to metastasis-associated macrophage polarization. Cell death and differentiation 2014 — PMC4085533.
- Proinflammatory protein CARD9 is essential for infiltration of monocytic fibroblast precursors and cardiac fibrosis caused by Angiotensin II infusion. American journal of hypertension 2011 — PMC3139445.
- Caspase Recruitment Domain-Containing Protein 9 Expression is a Novel Prognostic Factor for Lung Adenocarcinoma. OncoTargets and therapy 2020 — PMC7498929.
- PubMed PMID:11053425 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CARD9 (Caspase recruitment domain-containing protein 9). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01410-1). Validated IHC antibody: A01410-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Elongated or late spermatids); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A01410-1); 2. Fixation: Image fixative and duration unreported (datasheet A01410-1); 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 A01410-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01410-1); 8. Primary antibody: Rabbit anti-CARD9, 0.5-1μg/ml (datasheet A01410-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01410-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining was disregarded (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage needs sequence review (UniProt); Regulation: Expression restricted to phagocyte populations (UniProt). Evidence: UniProt Q9H257, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in THP-1: 63.3 nTPM, Datasheet A01410-1, PMC4085533, PMC3139445. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CARD9/immunohistochemistry-ihc-guide.