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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 reported cytoplasmic, nuclear and membrane staining pattern (HPA tissue IHC). Start catalog antibody A13067 at 1:50–1:200 (datasheet), with glandular or hematopoietic cells as positive tissue controls (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 and endothelial cells; cytoplasmic, nuclear, membrane (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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Medium antibody–RNA concordance; verify staining (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; chain spans residues 2–1037 (UniProt) |
Section 1
Recommended CARD6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by one published CARD6 immunohistochemistry protocol (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; it matches the published method (PMC4792137) and this page’s retrieval setting (page specification).
Section 2
What Is the Expected CARD6 Staining Pattern?
CARD6 staining in paraffin sections may appear in the cytoplasm, nucleus, or at cell membranes (HPA: tissue IHC profile). High staining is reported in glandular, hematopoietic, respiratory epithelial, and endothelial cells in the listed tissues (HPA: tissue IHC). CARD6 has no annotated transmembrane segment (UniProt Q9BX69 topology). The tissue profile is Approved, with medium consistency between antibody staining and RNA data; external verification is pending (HPA: reliability).
| Distinct cellular chromogen in glandular cells of adrenal gland, appendix, cervix, or duodenum. | These are reported High staining combinations (HPA: tissue IHC). Assess the named cell population; the tissue name alone does not identify which cells carry the signal. |
| Cytoplasmic, nuclear, or membrane-associated staining in an expected cell population. | All three compartments fit the reported broad pattern (HPA: tissue IHC profile). A membrane-associated signal does not establish that CARD6 spans the membrane (UniProt Q9BX69 topology). |
| Chromogen confined to tissue spaces, deposits, or a cell population outside the reported examples. | Treat this as uncertain rather than confirmed CARD6 staining. Compare cell morphology and a negative control; nonspecific antibody binding or endogenous detection activity can mimic signal (standard IHC practice). |
| Weak, diffuse color across many structures, obscuring cell boundaries. | The distribution is difficult to score against the reported cellular pattern (HPA: tissue IHC profile). Background can arise from detection or blocking conditions (standard IHC practice). |
| No discernible cellular signal in a section containing a reported High staining population. | This conflicts with the reference observation for that cell population (HPA: tissue IHC). Check slide quality, assay controls, and detection before interpreting the sample as CARD6 negative (standard IHC practice). |
💡Expected CARD6 appearanceCall a positive result when distinct cytoplasmic, nuclear, or membrane-associated chromogen is present in the reported High staining cell population (HPA: tissue IHC); diffuse color without identifiable positive cells is unconvincing (standard IHC practice).
| Reference cell population | High staining is reported in glandular, hematopoietic, respiratory epithelial, and endothelial populations in specified tissues (HPA: tissue IHC); select the population when scoring. |
| Compartment breadth | The reference spans cytoplasm, nucleus, and membrane (HPA: tissue IHC profile); a single compartment alone is insufficient to declare every other pattern erroneous. |
| Antibody evidence | The tissue profile is Approved with medium RNA–staining consistency and pending external verification (HPA: reliability). HPA041933 is IHC Approved, with no Enhanced claim supplied (HPA: antibodies). |
| Tissue distribution | RNA has low tissue specificity (HPA: tissue IHC). The supplied IHC list identifies High examples but supplies no negative or low staining tissue reference (HPA: tissue IHC). |
| Topology and processing | No transmembrane segment or signal peptide is annotated (UniProt Q9BX69 topology and processing). These annotations alone cannot assign a precise IHC compartment or explain every membrane-associated signal. |
| Fixation and retrieval evidence | Target-specific fixation sensitivity and retrieval conditions are unreported in the supplied sources (HPA: tissue IHC; UniProt Q9BX69). Record retrieval settings as assay conditions, without attributing their effect to CARD6. |
| ICC/IF evidence | No main subcellular location or ICC/IF image-bearing cell line is supplied (HPA: subcellular). The tissue IHC pattern therefore does not validate an ICC/IF staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a reported High staining population. | A failed detection step, unsuitable assay setting, or poor section quality is possible (standard IHC practice); the reference reports High staining for that population (HPA: tissue IHC). | Review section morphology and positive-control performance; check antibody, retrieval, detection, and counterstain records before calling the specimen negative (standard IHC practice). |
| Color is widespread and lacks clear cellular boundaries. | Nonspecific binding, residual detection activity, or excessive chromogen development may contribute (standard IHC practice). This is hard to reconcile with a scorable cellular pattern (HPA: tissue IHC profile). | Compare a negative control, review blocking and development conditions, and score only identifiable cells with signal above background (standard IHC practice). |
| Only extracellular material or tissue edges stain. | Deposits or edge-related staining may mimic a positive result (standard IHC practice); these sites are outside the reported cellular profile (HPA: tissue IHC profile). | Inspect adjacent morphology and a negative control; repeat with consistent section handling if the pattern persists (standard IHC practice). |
| Staining appears in a cell type outside the listed High examples. | The supplied list does not establish that other cell types are negative (HPA: tissue IHC); nonspecific staining remains possible (standard IHC practice). | Describe the observed cell type and compartment, check controls, and avoid classifying it as validated CARD6 staining from this reference alone (HPA: tissue IHC). |
| Nuclear or membrane-associated staining seems unexpected. | Both are included in the reported tissue profile (HPA: tissue IHC). UniProt provides no subcellular annotation and reports no transmembrane segment (UniProt Q9BX69). | Assess whether staining is cellular and control-supported; do not reject it solely for its compartment or infer membrane insertion from its appearance (HPA: tissue IHC; UniProt Q9BX69 topology). |
| Can the IHC appearance define an ICC/IF positive pattern? | No ICC/IF images or main subcellular location are supplied (HPA: subcellular); the available antibody status is IHC Approved (HPA: antibodies). | Treat ICC/IF localization as unestablished here and use the separate IF/ICC guide for that application (HPA: subcellular). |
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 assess staining in glandular cells (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, and assess adjacent nonglandular cells for background without treating them as proven CARD6-negative cells (HPA: no negative rows; CARD6 detected in all 45 scored tissues).
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: Include a no-primary, secondary-only control; use nonimmune immunoglobulin matched to the primary antibody’s host species and isotype as the isotype control, and use CARD6 knockout material or a validated peptide block as a biological specificity control (standard IHC practice). For appendix, check endogenous peroxidase activity in inflammatory cells before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: Paraffin-section IHC is documented for the catalog antibody at 1:100 in human breast carcinoma, but the caption does not report a fixative (selected A13067 tissue-IHC caption). The supplied evidence reports no CARD6-specific fixation window or retrieval dependency; establish antigen retrieval empirically (supplied target/application evidence; standard IHC practice). It also does not establish whether frozen-section IHC or IF is easier, and the appendix’s inflammatory cells can contribute endogenous peroxidase signal (supplied target/application evidence; 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
Troubleshoot CARD6 staining in paraffin sections by checking retrieval, cell identity and compartment before interpreting chromogenic signal.
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 minutes (page retrieval setting). Allow sections to cool in buffer, then compare stained sections with a matched primary-antibody omission control (standard IHC practice). If signal remains weak, test a small range of heating times on adjacent sections while holding the antibody dilution and detection conditions constant (standard IHC practice). The selected CARD6 image shows staining in paraffin-embedded human breast carcinoma at 1:100, but its caption gives no retrieval method or fixative (A13067 caption).
Could fixation explain inconsistent CARD6 staining across my paraffin sections?
CARD6-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (A13067 caption; UniProt Q9BX69). The selected image documents paraffin-embedded human breast carcinoma but does not state its fixative, so it cannot establish a CARD6 fixation condition (A13067 caption). Record each specimen’s fixative and duration, and compare similarly processed sections with the same retrieval and antibody conditions (standard IHC practice). If staining varies, include a consistently processed reference section in each run and assess morphology alongside signal before attributing the difference to CARD6 expression (standard IHC practice).
Which compartments should count as plausible CARD6 staining?
Assess cytoplasmic, nuclear and membrane-associated signal, since all three appear in the reported tissue IHC profile (HPA tissue IHC). UniProt does not annotate a CARD6 subcellular location, and its sequence has no transmembrane segment (UniProt Q9BX69). A membrane-like outline therefore warrants comparison with adjacent cells and controls before it is scored as specific staining (HPA tissue IHC; standard IHC practice). Record each compartment separately, compare the pattern across intact cells, and inspect the primary-antibody omission control; a single unexpected compartment should not define positivity by itself (standard IHC practice).
How can epitope location affect interpretation of CARD6 IHC?
CARD6 has a CARD domain at residues 3–94, while the supplied record lists no alternative isoforms (UniProt Q9BX69). Its annotated modifications include N-acetylalanine at residue 2 and phosphoserines at 154 and 985 (UniProt Q9BX69). The selected image caption does not identify the antibody epitope, so none of those features establishes whether a particular modification affects staining (A13067 caption; UniProt Q9BX69). Check the antibody’s documented immunogen or epitope before comparing protocols, and use adjacent sections with identical processing when assessing a suspected epitope-dependent difference (standard IHC practice).
How should I evaluate CARD6 by IF alongside the IHC result?
Treat IF as a separate assay: the supplied HPA subcellular record lists no CARD6 ICC/IF images for direct comparison (HPA subcellular). Multiplex CARD6 with a validated marker for the cell population being examined; reported high-staining examples include bronchial respiratory epithelial cells and colon endothelial cells (HPA tissue IHC; standard IF practice). Choose fluorophores after checking tissue autofluorescence in an unstained section, and include single-label controls to assess channel bleed-through (standard IF practice). Set permeabilisation according to whether the documented antibody epitope is accessible at the cell surface or requires intracellular access; the selected IHC caption does not specify that epitope (A13067 caption; standard IF practice).
What should I check when CARD6 chromogenic staining looks diffuse?
First inspect a primary-antibody omission control for detection-system staining, and examine a substrate-only control if endogenous enzyme activity is suspected (standard IHC practice). Use a peroxidase block before DAB development when the detection system is peroxidase based (standard IHC practice). Compare background in tissue folds, cut edges and damaged regions with intact central tissue, and review the counterstain to locate the signal within cells (standard IHC practice). The HPA profile includes cytoplasmic, nuclear and membrane expression, so diffuse color alone does not identify a CARD6-positive compartment without cell boundaries and appropriate controls (HPA tissue IHC; standard IHC practice).
How should I score CARD6 staining across heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since the reported CARD6 tissue IHC profile spans cytoplasm, nucleus and membrane (HPA tissue IHC). For intact cells, report the percentage positive and an H-score from intensity categories, applying one threshold across sections in the comparison (standard IHC practice). If counting positive cells, report density per mm² of viable tissue and exclude folds, necrosis and tissue-free areas from the denominator (standard IHC practice). Normalise comparisons to the same cell type, viable area, processing batch and exposure to detection reagents; document any compartment-specific score separately (standard IHC practice).
How can I distinguish CARD6 signal from artefact in a positive section?
Look for repeatable staining in intact, identifiable cells rather than signal confined to edges, folds or necrotic areas (standard IHC practice). HPA reports high staining in several defined populations, including appendix glandular cells and bone marrow hematopoietic cells, but assigns its tissue IHC profile Approved reliability with pending external verification (HPA tissue IHC). Check whether the stained cell type and compartment match the recorded field; cytoplasmic, nuclear and membrane patterns are reported, while UniProt provides no subcellular annotation (HPA tissue IHC; UniProt Q9BX69). Review primary-antibody omission and endogenous peroxidase controls before interpreting brown deposits as CARD6-specific signal (standard IHC practice).
Boster reagents
Best CARD6 / Caspase recruitment domain-containing protein 6 IHC Antibodies
The catalog lists human-reactive anti-CARD6 antibodies for IHC and IF; the supplied figure shows IHC in paraffin-embedded human breast carcinoma (catalog applications; A13067 image caption).
Real IHC data
Anti-CARD6 (A2) Antibody
Cat # A13067
A13067 will render with its IHC figure from paraffin-embedded human breast carcinoma at 1:100 (A13067 image caption). IF is listed for A13067, but no IF figure is supplied (A13067 catalog applications and image alts).
Which to pick: Choose A13067 for tissue IHC because its own figure documents paraffin-embedded human breast carcinoma; the fixative is unreported (A13067 image caption). For IF, both A13067 and A13067-1 list human reactivity and IF, but neither supplies an IF figure or explicit ICC validation (catalog applications, reactivity and image alts). Neither SKU lists cross-species reactivity, and clonality cannot be established for A13067-1 from the supplied catalog data (catalog reactivity and clone fields).
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 paraffin sections. (standard IHC practice; not target-specific); Caveat: Medium antibody–RNA concordance; verify staining (HPA tissue IHC); Isoform / epitope: No annotated isoforms; chain spans residues 2–1037 (UniProt); Regulation: No expression regulator annotated (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.