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CARD6 / Caspase recruitment domain-containing protein 6 · IHC design guide
Design Immunohistochemistry for CARD6
Plan chromogenic CARD6 IHC on paraffin sections using the catalog antibody A13067 at 1:50–1:200 (datasheet). Compare staining with HPA high-staining glandular cells and assess cytoplasmic, nuclear and membrane signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CARD6 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, nuclear and membrane staining (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic, nuclear and membrane staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A13067); verify before use. | |
| Caveat | Staining and RNA show medium consistency; verify specificity (HPA tissue IHC) | |
| Regulation | Low tissue specificity; stimulus regulation unreported (HPA tissue IHC; UniProt) | |
| Isoform / epitope | No isoforms or transmembrane segment annotated (UniProt) |
Section 1
Recommended CARD6 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published CARD6 method using paraffin sections (PMC4792137).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A13067) |
| Fixation | Image fixative and duration unreported (datasheet A13067); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CARD6, 1:50-1:200 (datasheet A13067) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CARD6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic, nuclear and membrane expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval for 20 min, as used in the published CARD6 method (PMC4792137).
Section 2
What Is the Expected CARD6 Staining Pattern?
In paraffin-section IHC, expect CARD6 staining in cytoplasm, nuclei, or at cell membranes; HPA reports a broad pattern and does not assign a single required compartment (HPA tissue IHC). High staining is reported in specified glandular, hematopoietic, respiratory epithelial, and endothelial cells (HPA: High). Interpret this as an expected pattern, not definitive target verification: HPA rates the IHC evidence Approved, with medium staining–RNA consistency and external verification pending (HPA tissue IHC). CARD6 has no annotated transmembrane segment (UniProt Q9BX69 topology).
| Cell-associated chromogen appears in cytoplasm, nuclei, or at membranes of HPA-listed cell types. | This fits the reported broad CARD6 pattern (HPA tissue IHC). Compare staining within identified cells; compartment alone cannot establish antibody specificity. High staining in those cells is an HPA observation, not a required score for every section (HPA: High). |
| Signal is confined to luminal material, extracellular matrix, debris, or a sharp tissue edge. | These sites fall outside HPA's reported cell-associated cytoplasmic, nuclear, and membrane pattern (HPA tissue IHC). Suspect deposition or another staining artefact; inspect morphology and controls before scoring cells. Nuclear or membrane staining alone is not a wrong-compartment finding here (HPA tissue IHC). |
| Strong staining appears mainly in cells other than those identified as High by HPA. | Check cell identity and the detection controls before attributing it to CARD6. Cross-reactivity or endogenous detection activity are possible explanations (general IHC practice). HPA reports low tissue RNA specificity, so staining in another cell type cannot by itself prove cross-reactivity (HPA tissue IHC). |
| A uniform haze covers several cell types, stroma, or the whole section. | A field-wide signal obscures the cell-associated pattern needed for interpretation (HPA tissue IHC). Nonspecific binding, residual reagent, or chromogen development may contribute (general IHC practice). Treat intensity scores as unreliable until background is controlled. |
| There is no detectable signal in a section expected to contain HPA-listed High cells. | First verify that the relevant cells are present and preserved; examples include adrenal glandular cells and bone-marrow hematopoietic cells (HPA: High). A blank result then warrants a check of antibody, retrieval, and detection conditions (general IHC practice). It does not establish absence of CARD6. |
💡Expected CARD6 appearanceA convincing positive is cell-associated cytoplasmic, nuclear, or membrane chromogen in morphologically identified HPA-listed cells, potentially strong where HPA scores staining High; uniform haze or deposits outside cells are suspect (HPA tissue IHC; general IHC practice).
| Compartment and topology | HPA reports cytoplasmic, nuclear, and membrane staining (HPA tissue IHC). CARD6 lacks an annotated transmembrane segment (UniProt Q9BX69 topology); membrane-associated staining should therefore be described as an observed pattern, not evidence that CARD6 spans the membrane. |
| Choice of positive tissue and cell type | HPA lists High staining in glandular cells of adrenal gland, appendix, cervix, and duodenum; hematopoietic cells of bone marrow; respiratory epithelial cells of bronchus; and endothelial cells of cerebral cortex and colon (HPA: High). Select a listed cell population that can be identified on the section. |
| Strength of antibody evidence | HPA lists antibody HPA041933 as a rabbit polyclonal with IHC Approved status, and describes medium staining–RNA consistency with external verification pending (HPA antibodies; HPA tissue IHC). The supplied record does not report IHC Enhanced validation (HPA antibodies). Interpret agreement with the pattern as supportive rather than conclusive. |
| Can this IHC pattern be used as an IF/ICC expectation? | HPA provides no main ICC-IF location and lists no cell lines with ICC-IF images for CARD6 (HPA subcellular). The tissue IHC pattern can guide what to inspect, but it does not establish an IF/ICC localisation result. This section supplies no IF/ICC protocol option. |
| Target-specific retrieval or processing effect | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| An HPA-listed High cell population is present, but the section is blank. | The primary antibody, retrieval, or detection sequence may have failed (general IHC practice); a blank section alone does not identify which step failed. | Confirm cell identity, then check a concurrent positive section, reagent application, retrieval conditions, and detection steps (general IHC practice). Use an HPA-listed High cell population as the expected reference (HPA: High). |
| Most of the section shows weak, diffuse brown haze. | Nonspecific binding or excess chromogen development can obscure cell boundaries (general IHC practice). | Review blocking, antibody dilution, wash steps, and development time; score only interpretable cell-associated signal (general IHC practice; HPA tissue IHC pattern). No CARD6-specific working dilution is supplied. |
| Brown signal persists in a control run without primary antibody. | Endogenous detection activity or a detection-system artefact may be present (general IHC practice). | Investigate the detection reagents and, where relevant, endogenous enzyme blocking before assigning the signal to CARD6 (general IHC practice). |
| Signal concentrates at cut edges, folds, debris, or luminal material. | Section geometry or precipitated detection product can mimic a positive cell pattern (general IHC practice). | Inspect an intact area and compare with the control section; exclude deposits outside identifiable cells from scoring (general IHC practice; HPA tissue IHC pattern). |
| Strong staining occurs in an unexpected cell population. | Cell identification, cross-reactivity, or endogenous activity may explain the result (general IHC practice). HPA's low tissue RNA specificity does not make an unlisted population definitively negative (HPA tissue IHC). | Recheck morphology and detection controls, then report the observed cell type and compartment without claiming CARD6 specificity from staining alone (general IHC practice; HPA tissue IHC). |
| A reader expects only cytoplasmic staining or treats all membrane staining as invalid. | That rule conflicts with HPA's general cytoplasmic, nuclear, and membrane profile (HPA tissue IHC). The absence of a transmembrane segment does not resolve the observed membrane pattern (UniProt Q9BX69 topology). | Record compartment and cell type separately, compare with HPA-listed High cells, and qualify uncertain localisation rather than discarding it by compartment alone (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CARD6 staining
A13067 · IHCImmunohistochemistry (IHC) analyzes of CARD6 (A2) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132357-CARD6/tissue/cerebral+cortex#imid_21189619
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132357-CARD6/tissue/colon#imid_21189545
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132357-CARD6/tissue/liver#imid_21189554
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132357-CARD6/tissue/kidney#imid_21189563
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CARD6 IHC & IF
🧪Run appendix first and score its glandular cells as the expected positive population (HPA: High in appendix glandular cells). HPA detects CARD6 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls to define background in adjacent nonglandular cells, without assuming those cells are CARD6-negative (HPA: no negative rows; standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CARD6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CARD6; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: On adjacent sections, use a no-primary, secondary-only control and a control immunoglobulin matched to the primary antibody’s host species and clonality; confirm specificity with CARD6 knockout tissue or, if the immunizing peptide is available, a peptide-block control (standard IHC practice). Quench endogenous peroxidase for chromogenic detection, particularly when evaluating inflammatory cells in appendix (standard IHC practice).
⚠️Feasibility: The selected A13067 caption shows CARD6 staining in a paraffin-embedded breast carcinoma section at 1:100, but does not report the fixative; a CARD6-specific fixation window or fixation effect is unreported (selected-SKU tissue-IHC caption). CARD6-specific retrieval requirements are unreported, so optimize antigen retrieval for the paraffin-section assay (standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier; in appendix, inflammatory-cell peroxidase can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for CARD6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CARD6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CARD6 IHC Tips
Use compartment, cell type, and section controls to interpret CARD6 staining in paraffin sections; its tissue IHC evidence does not establish a target-specific fixation response.
How should I retrieve CARD6 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting: cytoplasmic/membrane antigen rule). Apply the same cooling and wash steps to test and control sections so staining differences remain interpretable (standard IHC practice). The selected antibody image shows CARD6 staining in paraffin-embedded human breast carcinoma at 1:100, but its caption does not report retrieval conditions or fixative (A13067 caption). If signal remains weak, compare a carefully controlled alternative retrieval condition only after confirming section adhesion, antibody incubation, and detection performance (standard IHC practice). Excessive retrieval can damage morphology and complicate compartment scoring (standard IHC practice).
Can fixation explain inconsistent CARD6 staining between paraffin blocks?
CARD6-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not identify a fixative (A13067 caption). Record each block’s documented fixation conditions, then compare sections using matched retrieval, antibody incubation, and chromogenic development (standard IHC practice). For a new preparation, use a consistently applied routine tissue fixation workflow and preserve morphology before adjusting retrieval (standard IHC practice). Weak staining in one block alone cannot establish fixation damage, because processing and detection can also vary (standard IHC practice). Assess a concurrently stained positive control and tissue morphology before interpreting the difference as biological (standard IHC practice).
Which cellular compartments should count as plausible CARD6 staining?
Evaluate cytoplasmic, nuclear, and membrane-associated signal separately because the tissue IHC profile reports all three patterns (HPA tissue IHC profile). CARD6 has no annotated transmembrane segment, while UniProt supplies no subcellular location, so a membrane-like outline is not proof of an integral membrane pool (UniProt Q9BX69 topology and subcellular record). Compare the apparent compartment with intact cell boundaries and the counterstained nucleus on the same section (standard IHC practice). HPA reports high staining in adrenal glandular cells and bone-marrow hematopoietic cells, among other sites, offering cell-type reference patterns (HPA tissue IHC). Score unexpected sharply outlined or diffuse extracellular deposits separately until controls establish their specificity (standard IHC practice).
How can I assess whether the antibody recognises the intended CARD6 epitope?
The supplied record lists no alternative CARD6 isoforms and places the CARD domain at residues 3–94 (UniProt Q9BX69 record). Its reported modified residues include N-acetylalanine at 2 and phosphoserines at 154 and 985; their effects on this antibody are unknown because its epitope is unspecified here (UniProt Q9BX69 record; supplied antibody evidence). Request the immunogen or epitope information before attributing compartment differences to epitope masking or a modification (standard IHC practice). Compare staining with an independent antibody to a documented different epitope, if available, and include an appropriate negative control (standard IHC practice). Neither staining intensity nor location alone identifies a particular modified CARD6 population (standard IHC interpretation).
How should an IF follow-up test the cell types seen by CARD6 chromogenic IHC?
Treat IF as a separate validation experiment because the supplied selected-antibody image documents paraffin-section IHC at 1:100, not IF conditions (A13067 caption). Multiplex CARD6 with a validated marker for the cell type under study, such as an endothelial marker when evaluating the reported high endothelial staining in cerebral cortex (HPA tissue IHC; standard IF practice). Select fluorophores after checking the tissue’s autofluorescence and image single-stain controls to assess bleed-through (standard IF practice). CARD6 has no annotated transmembrane segment and no assigned subcellular location, so choose permeabilisation after establishing whether the tested epitope requires intracellular access (UniProt Q9BX69 topology and subcellular record; standard IF practice).
What should I check when CARD6 chromogenic staining looks widespread or patchy?
Inspect unstained and no-primary sections for pigment, endogenous peroxidase activity, and nonspecific chromogen deposits before assigning CARD6 positivity (standard chromogenic IHC practice). Apply a peroxidase block and appropriate protein blocking as workflow controls, then keep DAB development consistent across comparison sections (standard chromogenic IHC practice). HPA describes general cytoplasmic, nuclear, and membrane expression, so widespread staining alone cannot distinguish signal from background (HPA tissue IHC profile). Check whether intense colour follows section edges, folds, damaged regions, or necrosis rather than intact cells (standard IHC practice). Reassess antibody concentration using the selected paraffin-section image’s 1:100 condition as a documented reference, not a guaranteed optimum (A13067 caption).
How should I score CARD6 staining across different tissues or cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the target cell population and compartment before scoring, since HPA reports cytoplasmic, nuclear, and membrane patterns across tissues (HPA tissue IHC profile). For chromogenic sections, record the percentage of positive intact target cells and an H-score using intensity grades 0–3, yielding a score of 0–300 (standard IHC scoring practice). For scattered populations, report positive-cell density per mm² of viable tissue or the percentage among the corresponding cell type (standard IHC quantification practice). Normalize comparisons to evaluated viable area or target-cell count, and hold threshold, retrieval, and DAB development constant (standard IHC practice). Exclude folds, necrosis, and edge artefacts consistently (standard IHC practice).
How can I separate convincing CARD6 positivity from a staining artefact?
Call a pattern convincing when signal localizes to intact cells, repeats across comparable sections, and exceeds the appropriate negative-control signal (standard IHC interpretation). HPA reports high staining in several glandular, hematopoietic, epithelial, and endothelial populations, but its tissue IHC reliability is Approved with medium RNA concordance and pending external verification (HPA tissue IHC). Treat a compartment or cell population outside those observations as a result requiring confirmation, since UniProt does not annotate CARD6 subcellular location (HPA tissue IHC; UniProt Q9BX69 subcellular record). Discount edge staining, necrotic deposits, and residual endogenous-enzyme colour when they also appear in controls (standard chromogenic IHC practice). Corroborate consequential claims with an independent method (standard IHC practice).
Boster reagents
Best CARD6 / Caspase recruitment domain-containing protein 6 IHC Antibodies
The catalog lists two human-reactive CARD6 antibodies for IHC and IF (catalog: applications and reactivity). A13067 has an IHC image from a paraffin-embedded human breast carcinoma section (catalog image caption).
Real IHC data
Anti-CARD6 (A2) Antibody
Cat # A13067
A13067 is the only SKU with a rendered card; its image shows IHC on paraffin-embedded human breast carcinoma at 1:100 (catalog image caption). It also lists IF and human reactivity, but has no IF image (catalog: applications, reactivity, image alts).
Which to pick: Choose A13067 for tissue IHC because its own image documents a paraffin-embedded human section; the fixative is unreported (catalog image caption). For IF, both A13067 and A13067-1 list the application, but neither provides an IF image or lists ICC, so there is no documented basis to prefer one for IF/ICC (catalog: applications and image alts). Neither has documented cross-species reactivity: both list Human only, and neither reports a clone (catalog: reactivity and clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BX69 (CARD6_HUMAN, Caspase recruitment domain-containing protein 6).
- Human Protein Atlas. CARD6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CARD6 subcellular location (ICC-IF): Highest expression in HUVEC/TERT2: 31.4 nTPM.
- Human Protein Atlas. CARD6 antibody validation summary (1 antibodies).
- NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis. Scientific reports 2016 — PMC4792137.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:22814378 — UniProt-cited evidence.
- PubMed PMID:23186163 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CARD6 (Caspase recruitment domain-containing protein 6). Expected localisation Cytoplasmic, nuclear and membrane staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A13067 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A13067); 2. Fixation: Image fixative and duration unreported (datasheet A13067); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CARD6, 1:50-1:200 (datasheet A13067); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A13067); verify before use.; Caveat: Staining and RNA show medium consistency; verify specificity (HPA tissue IHC); Isoform / epitope: No isoforms or transmembrane segment annotated (UniProt); Regulation: Low tissue specificity; stimulus regulation unreported (HPA tissue IHC; UniProt). Evidence: UniProt Q9BX69, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in HUVEC/TERT2: 31.4 nTPM, Datasheet A13067, PMC4792137. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CARD6/immunohistochemistry-ihc-guide.