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CARF / Calcium-responsive transcription factor · IHC design guide
Design Immunohistochemistry for CARF
Plan CARF staining in paraffin sections using its observed nuclear and cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation, controls and scoring, with high-staining reference cells including cerebellar Purkinje cells and pancreatic exocrine glandular cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CARF 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 | Nuclear and cytoplasmic tissue staining (HPA tissue IHC); nuclear location (UniProt) | |
| Staining pattern | Positive tissue cells show nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+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 | Purkinje cells stain high; hippocampal neurons stain low (HPA tissue IHC) | |
| Regulation | CARF expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; no extracellular/cytoplasmic split; epitope unknown (UniProt; datasheet) |
Section 1
Recommended CARF IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with 2 published CARF IHC workflows: mouse paraffin sections (PMC12040762) and HCC tissues (PMC5348329).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A30570) |
| Fixation | Image fixative and duration unreported (datasheet A30570); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-CARF, 1:100 - 1:300 (datasheet A30570) |
| 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 | CARF-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval); citrate pH 6 is a published alternative (PMC12040762).
Section 2
What Is the Expected CARF Staining Pattern?
In paraffin-section IHC, expect nuclear and cytoplasmic CARF staining in selected epithelial, glandular, neuronal and decidual cells (HPA tissue IHC). UniProt places CARF in the nucleus and annotates no transmembrane segment (UniProt Q8N187). HPA rates its tissue staining Approved, with medium consistency against RNA data and external verification pending (HPA tissue IHC). Interpret the stained cell type and compartment together.
| Nuclear and cytoplasmic signal in pancreatic exocrine glandular cells or cerebellar Purkinje cells. | These are high-staining cell populations (HPA tissue IHC). Score signal in the identified cells, using tissue morphology and the counterstain to distinguish nuclei from adjacent cytoplasm (general IHC practice). |
| Only sharp cell-surface staining, with no convincing nuclear or cytoplasmic component. | That distribution does not match the reported IHC profile (HPA tissue IHC) or nuclear annotation and lack of a transmembrane segment (UniProt Q8N187). Check morphology, detection background and antibody specificity before calling it CARF. |
| Strong staining is concentrated in an unexpected cell population. | Compare with the named HPA cell populations; unexpected staining can reflect cross-reactivity or endogenous chromogenic activity (general IHC practice). It is not automatically false: HPA reports low tissue specificity at the RNA level and supplies no validated negative tissue (HPA tissue IHC). |
| Diffuse chromogen covers many cells or tissue spaces without clear cellular boundaries. | Treat the pattern as background until a no-primary control and the tissue morphology support specific staining (general IHC practice). HPA's nuclear and cytoplasmic description refers to cellular localisation, not a diffuse field-wide deposit (HPA tissue IHC). |
| No signal appears in a tissue with a reported high-staining cell population. | First confirm that the relevant cells are present; for example, pancreatic exocrine glandular cells are reported High (HPA tissue IHC). Then check the positive control, retrieval, primary-antibody conditions and detection reagents (general IHC practice). |
💡Expected CARF appearanceCall a positive result when identifiable cells show nuclear and cytoplasmic chromogen, especially a reported High population such as Purkinje cells (HPA tissue IHC); isolated membrane staining or diffuse deposit warrants investigation (general IHC practice).
| Cell population and tissue choice | HPA reports High staining in eight listed cell populations, including respiratory epithelium and decidual cells, but Low staining in six listed populations, including hippocampal neurons (HPA tissue IHC). Choose controls by cell type, not tissue name alone (general IHC practice). |
| Antibody validation | HPA034557 is Approved for IHC; HPA043191 has no IHC status in this record (HPA antibodies). The tissue profile is Approved with medium RNA agreement and pending external verification (HPA tissue IHC), so review discordant staining cautiously. |
| Sequence and topology | CARF has three annotated isoforms, one 1–725 chain, no annotated glycosylation sites and no transmembrane segment (UniProt Q8N187). The payload gives no antibody epitope, so it cannot establish which isoforms an IHC antibody detects. |
| Target-specific fixation sensitivity | No fixation-effect evidence is supplied for CARF (UniProt Q8N187; HPA tissue IHC). If staining fails, assess retrieval and processing against controls as general IHC troubleshooting, without attributing the result to a proven CARF-specific masking effect. |
| How should IF/ICC be interpreted? | HPA reports mainly vesicular localisation, with additional nucleoli fibrillar-center and cytosolic signal in ICC-IF (HPA subcellular). That observation comes from a different application; use the IHC tissue profile to judge paraffin-section chromogenic staining. |
| Chromogenic detection | Endogenous enzyme activity or insufficient blocking can imitate antibody signal (general IHC practice). A no-primary control helps identify detection-derived deposit; it does not establish CARF expression in a particular cell. |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported High population stains weakly or not at all. | The relevant cells may be absent from the section, or the IHC workflow may have lost sensitivity (general IHC practice). | Confirm cell identity and run a positive tissue control alongside the section; then review retrieval, primary-antibody conditions and detection (general IHC practice). HPA's High designation is observational, not a guaranteed result in every specimen (HPA tissue IHC). |
| Chromogen appears throughout the section, including areas without identifiable target cells. | Nonspecific reagent binding or endogenous detection activity is possible (general IHC practice). | Inspect a no-primary control, blocking and washing; score only cell-associated staining with discernible morphology (general IHC practice). |
| Staining appears exclusively along cell borders. | The pattern conflicts with nuclear and cytoplasmic tissue staining (HPA tissue IHC) and the lack of a transmembrane segment (UniProt Q8N187). | Check for edge artifact and detection background, then reassess with an IHC-validated antibody and appropriate controls (general IHC practice; HPA antibodies: HPA034557 Approved). |
| Unexpected cell types stain strongly while the expected population does not. | Cross-reactivity, endogenous activity or mistaken cell identification are possibilities (general IHC practice); HPA provides no validated negative tissue in this payload (HPA tissue IHC). | Recheck morphology and the no-primary control; compare the same named cell population in a reported High tissue before interpreting the unexpected signal (HPA tissue IHC; general IHC practice). |
| An IHC section lacks the vesicular pattern described for ICC-IF. | The vesicular observation comes from HPA's ICC-IF record, whereas its tissue IHC profile is nuclear and cytoplasmic (HPA subcellular; HPA tissue IHC). | Judge the chromogenic section by the IHC cell and compartment profile; do not require an ICC-IF pattern in paraffin tissue. |
| A Low population stains faintly beside a strongly stained control population. | HPA reports different staining levels by cell population, including Low in hippocampal neurons and High in Purkinje cells (HPA tissue IHC). | Score each identified population separately and check that the stronger signal remains cell-associated; avoid treating faint staining alone as assay failure (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CARF staining
A30570 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using ALS2CR8 Antibody. The picture on the right is blocked
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000138380-CARF/tissue/cerebral+cortex#imid_8179803
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000138380-CARF/tissue/colon#imid_8179727
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000138380-CARF/tissue/liver#imid_8179712
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000138380-CARF/tissue/kidney#imid_8179713
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA034557; green = CARF, blue = nucleus, red = microtubules.
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA034557; green = CARF, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CARF IHC & IF
🧪Run appendix first; its glandular cells should stain strongly (HPA: High in appendix glandular cells). HPA detects CARF in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; cells on the positive slide without specific signal should remain at background, but no cell type there is established as CARF-negative (HPA: no negative tissue rows).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: CARF 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 ICC-IF images show CARF in SH-SY5Y, SiHa, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, clonality and concentration; use peptide blocking as a biological specificity check (selected A30570 tissue-IHC caption: signal blocked with synthesized peptide). For appendix chromogenic IHC, quench endogenous peroxidase and check for residual background in the controls (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected A30570 paraffin-section caption does not state a fixative (selected A30570 tissue-IHC caption: fixative unreported). CARF-specific antigen retrieval dependence and whether frozen sections or IF are easier are unreported in the supplied evidence. In appendix, endogenous peroxidase can produce chromogenic background, so assess it with the controls (standard IHC practice).
HPA tissue IHC evidence for CARF
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CARF is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CARF IHC Tips
Troubleshoot CARF staining in paraffin sections by checking retrieval, compartment, controls and scoring before interpreting chromogenic signal.
What retrieval should I use when CARF staining is weak in paraffin sections?
Use Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min on paraffin sections (page retrieval setting). Keep section thickness, heating vessel and cooling time consistent across test and control slides so a change in staining can be attributed to the retrieval adjustment (standard IHC practice). If staining remains weak, compare a shorter exposure with the same buffer before testing citrate pH 6.0 as a fallback (standard IHC practice). Include a section with expected CARF staining, such as bronchial respiratory epithelium (HPA: High in respiratory epithelial cells). Score nuclear and cytoplasmic signal separately because both appear in tissue staining (HPA: nuclear and cytoplasmic expression).
Could fixation explain weak or uneven CARF staining?
The selected paraffin-section caption does not state a fixative, so CARF-specific sensitivity to fixation is unknown (A30570 tissue-IHC caption). Record the fixative, time in fixative and processing history for each block before comparing staining across specimens (standard IHC practice). On sections with known processing histories, run the same retrieval and detection conditions alongside a positive control to assess whether the problem tracks with a block (standard IHC practice). Check for patchy preservation, section lifting and strong staining confined to exposed tissue edges, then repeat on a better-preserved section if available (standard IHC practice). Do not assign a CARF-specific fixation effect from these observations alone.
Should CARF staining be nuclear, cytoplasmic or vesicular?
Assess nuclear and cytoplasmic staining as separate patterns in chromogenic tissue sections (HPA: nuclear and cytoplasmic expression). UniProt lists CARF in the nucleus, while cell imaging places it mainly in vesicles and additionally in the nucleolar fibrillar center and cytosol (UniProt Q8N187 subcellular location; HPA subcellular). These sources support inspecting more than one compartment, but a diffuse stain alone cannot establish which structure contains CARF. Compare compartment patterns in the same cell type across test and positive-control sections, using a nuclear counterstain to define nuclei (standard IHC practice). Respiratory epithelial cells in bronchus offer a documented high-staining comparison (HPA: High in bronchial respiratory epithelial cells).
Can this stain distinguish CARF isoforms or identify a masked epitope?
CARF has 3 listed isoforms, but the supplied antibody information does not map its epitope to an isoform (UniProt Q8N187 isoforms; A30570 tissue-IHC caption). Therefore, score the chromogenic signal as CARF immunoreactivity rather than assigning it to isoform 1, 2 or 3. The selected paraffin-section image includes a synthesized-peptide blocking comparison, which helps assess antibody-dependent staining in that image (A30570 tissue-IHC caption). Peptide competition does not establish which isoform contributes to a stained compartment or rule out every cross-reactive target (standard IHC interpretation). If isoform attribution matters, obtain an epitope map and compare isoform-specific reference material before interpreting tissue differences.
How should I check CARF localisation by multiplex immunofluorescence?
Treat IF/ICC as a separate assay and first establish signal and controls with the intended specimen preparation (standard IF practice). In bronchial tissue, pair CARF with a respiratory epithelial marker, such as pan-cytokeratin, to identify the cell population reported as high staining (HPA: High in bronchial respiratory epithelial cells). Choose a far-red CARF fluorophore when tissue autofluorescence obscures shorter wavelengths, and inspect unstained and single-color controls before interpreting overlap (standard IF practice). CARF has no transmembrane segment and has reported nuclear, cytosolic and vesicular locations, so test gentle permeabilisation for intracellular access (UniProt Q8N187 topology and subcellular location; HPA subcellular). Compare permeabilised and untreated preparations because detergent can alter apparent punctate staining (standard IF practice).
How do I separate CARF signal from chromogenic background?
Start with a no-primary control and inspect whether the same deposits appear in the test section (standard IHC practice). Quench endogenous peroxidase before an HRP and DAB detection sequence, then keep chromogen development time matched across slides (standard IHC practice). If both controls and test sections darken, check blocking, secondary-antibody concentration and wash conditions before changing the CARF primary antibody (standard IHC practice). The selected paraffin-section image includes a synthesized-peptide blocking comparison, which provides an additional antibody-dependent staining check for that specimen (A30570 tissue-IHC caption). Read fine cellular staining separately from pigment, tissue folds, necrotic debris and staining at cut edges (standard IHC practice).
How should I score CARF staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue staining has been reported in both nuclei and cytoplasm (HPA: nuclear and cytoplasmic expression). For each compartment, record the percentage of positive target cells and an intensity score under fixed illumination and staining conditions (standard IHC practice). An H-score combines percentages at intensities 0–3 and spans 0–300; report the scoring thresholds and the number of cells assessed (standard IHC practice). Normalise positive-cell counts to all eligible cells of the same type, or stained-cell density to analysed tissue area in mm² (standard IHC practice). Exclude folds, necrosis and missing tissue consistently across sections (standard IHC practice).
When is a CARF-positive cell convincing rather than an artefact?
Look for reproducible cellular staining in the expected compartment and cell population across serial or replicate sections, with little corresponding signal in the no-primary control (standard IHC practice). Bronchial respiratory epithelial cells and cerebellar Purkinje cells are reported as high-staining examples, while hippocampal neuronal cells are reported as low (HPA tissue IHC). Nuclear and cytoplasmic tissue staining is reported, so cytoplasmic signal alone is not grounds for rejection (HPA: nuclear and cytoplasmic expression). Treat isolated edge staining, necrotic deposits and signal that persists without primary antibody as potential processing or endogenous-enzyme artefacts (standard IHC practice). Interpret unexpected patterns cautiously because the tissue staining assessment has medium consistency with RNA data and awaits external verification (HPA: Approved reliability description).
Boster reagents
Best CARF / Calcium-responsive transcription factor IHC Antibodies
The catalog lists A30570 for IHC, IF and ICC (catalog applications), with human lung carcinoma paraffin-section IHC data (catalog image caption) and Human, Mouse and Rat reactivity (catalog reactivity).
Real IHC data
Anti-ALS2CR8 Antibody
Cat # A30570
A30570 has an IHC image from a human lung carcinoma paraffin section, including a peptide-blocked comparison (catalog image caption). It is listed for IHC, IF and ICC in Human, Mouse and Rat (catalog applications; catalog reactivity); no IF image is supplied (catalog IF image alts).
Which to pick: Choose A30570 for paraffin-section tissue IHC because its own image caption documents that preparation (catalog image caption); the fixative is unreported (catalog image caption). Choose A30570 for IF/ICC because those applications are listed, while no IF image is supplied (catalog applications; catalog IF image alts). It is also the listed cross-species option for Human, Mouse and Rat, and is polyclonal (catalog reactivity; catalog dilution record).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N187 (CARTF_HUMAN, Calcium-responsive transcription factor).
- Human Protein Atlas. CARF tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CARF subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the nucleoli fibrillar center and cytosol..
- Human Protein Atlas. CARF antibody validation summary (2 antibodies).
- CARF enrichment promotes epithelial-mesenchymal transition via Wnt/β-catenin signaling: its clinical relevance and potential as a therapeutic target. Oncogenesis 2018 — PMC5945778.
- Fatty Acid Excess Dysregulates CARF to Initiate the Development of Hepatic Steatosis. Cells 2023 — PMC10093423.
- CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1. Acta biochimica et biophysica Sinica 2024 — PMC12040762.
- CARF activates beta-catenin/TCF signaling in the hepatocellular carcinoma. Oncotarget 2016 — PMC5348329.
- PubMed PMID:11586298 — UniProt-cited evidence.
- PubMed PMID:11832226 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CARF (Calcium-responsive transcription factor). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC); nuclear location (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A30570 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (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 lung carcinoma tissue; fixative not specified (datasheet A30570); 2. Fixation: Image fixative and duration unreported (datasheet A30570); 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 Tris-EDTA buffer, pH 9.0 , 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-CARF, 1:100 - 1:300 (datasheet A30570); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Purkinje cells stain high; hippocampal neurons stain low (HPA tissue IHC); Isoform / epitope: 3 isoforms; no extracellular/cytoplasmic split; epitope unknown (UniProt; datasheet); Regulation: CARF expression regulation is not annotated (UniProt). Evidence: UniProt Q8N187, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the nucleoli…, Datasheet A30570, PMC12040762, PMC5348329. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CARF/immunohistochemistry-ihc-guide.