This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CARF / Calcium-responsive transcription factor · IHC design guide
Design Immunohistochemistry for CARF
Plan CARF paraffin-section IHC using appendix glandular cells or cerebellar Purkinje cells as positive tissue references (HPA tissue IHC). This guide covers consistent fixation, a 1:100–1:300 catalog antibody dilution (datasheet), and interpretation of nuclear and cytoplasmic staining (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 staining observed; nucleus annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | High in appendix glandular cells; nuclear and cytoplasmic (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 sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | CARF expression regulation unannotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage unknown (UniProt) |
Section 1
Recommended CARF IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published CARF protocol for HCC tumor and paired normal tissue sections (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 rule; UniProt Q8N187: nucleus). The article leaves retrieval conditions unspecified (PMC5348329).
Section 2
What Is the Expected CARF Staining Pattern?
CARF is nuclear by UniProt (UniProt Q8N187: Nucleus; no transmembrane segment), while HPA tissue IHC reports nuclear and cytoplasmic expression (HPA: tissue IHC profile). Expect staining in glandular, respiratory epithelial, Purkinje, exocrine glandular, or decidual cells in the listed tissues (HPA: High in those cell types). HPA rates its tissue IHC Approved, with medium staining–RNA consistency and external verification pending (HPA: reliability).
| Distinct nuclear staining, with possible cytoplasmic staining, in the expected cells of a listed high-staining tissue. | This matches the HPA tissue IHC profile. Purkinje cells in cerebellum and exocrine glandular cells in pancreas are high-staining examples (HPA: High in both). Nuclear signal is consistent with UniProt localisation (UniProt Q8N187: Nucleus). |
| Signal is confined to cell borders or extracellular material, with no convincing intracellular pattern. | Treat this as a possible artefact and review morphology and controls (general IHC practice). HPA reports nuclear and cytoplasmic tissue staining, while UniProt annotates no transmembrane segment (HPA: tissue IHC profile; UniProt Q8N187: topology). |
| Strong staining appears mainly in an unexpected cell population while the expected cells show little signal. | Consider cross-reactivity or endogenous detection activity and check matched controls (general IHC practice). A low-staining population is not a proven negative: HPA reports Low staining in hippocampal neuronal cells and smooth muscle cells (HPA: Low). |
| Colour spreads diffusely across tissue or appears outside recognizable cells. | This limits confidence in compartment and cell-type scoring (general IHC practice). Reassess blocking, washing, chromogen development, and detection controls before calling it CARF staining (general IHC practice; HPA: nuclear and cytoplasmic tissue profile). |
| No signal is visible in a section containing a listed high-staining cell population. | First verify that the expected cells are present and that the run's detection controls worked (general IHC practice). Absence in, for example, prostate glandular cells conflicts with HPA's High observation, but one failed section cannot establish biological absence (HPA: High in prostate glandular cells). |
💡Expected CARF appearanceCall a positive result when recognisable expected cells show clear nuclear staining, with possible cytoplasmic staining, in a tissue where HPA reports High staining; diffuse extracellular colour or isolated cell-border staining is suspect (HPA: tissue IHC profile and High cell-type observations; UniProt Q8N187: Nucleus and no transmembrane segment; general IHC practice).
| Compartment and topology (UniProt Q8N187; HPA: tissue IHC profile) | UniProt places CARF in the nucleus and annotates no transmembrane segment; HPA tissue IHC also reports cytoplasmic expression. Score nuclear and cytoplasmic compartments separately when the section permits it (UniProt Q8N187: localisation and topology; HPA: tissue IHC profile; general IHC practice). |
| Cell-type context (HPA: High and Low tissue IHC observations) | HPA reports High staining in specified cell populations across eight listed tissues and Low staining in six others. Compare like cell types and tissue architecture; the HPA RNA label is Low tissue specificity, so an unlisted tissue is not an established negative (HPA: tissue IHC and RNA specificity; general IHC practice). |
| Isoforms and processing (UniProt Q8N187: isoforms and processing) | UniProt lists three isoforms and a single annotated chain spanning residues 1–725, with no signal peptide or propeptide. The payload gives no antibody epitope, so these annotations cannot predict which isoforms an IHC antibody detects (UniProt Q8N187: isoforms and processing). |
| Evidence and application (HPA: antibody validation and subcellular ICC-IF) | HPA034557 is Approved for IHC and ICC; HPA043191 is Approved for ICC but has no listed IHC status. HPA tissue IHC remains pending external verification, so ICC approval does not establish a second IHC confirmation (HPA: antibody statuses and tissue IHC reliability). |
| Situation | Likely cause | Next action |
|---|---|---|
| A listed high-staining tissue shows no CARF signal. | The expected cell population may be absent from the section, or the staining run may have failed (general IHC practice; HPA: High cell-type observations). | Confirm the named cells on morphology, inspect the run's positive detection control, and repeat with a documented IHC-validated antibody if needed (general IHC practice; HPA: HPA034557 IHC Approved). |
| The whole section has diffuse colour and compartments cannot be scored. | Background can arise from blocking, washing, chromogen development, or endogenous detection activity (general IHC practice). | Review reagent controls and the detection system, then optimise blocking, washes, and development using standard IHC controls (general IHC practice). Interpret only cell-associated signal that can be localized (HPA: nuclear and cytoplasmic tissue profile). |
| Staining is concentrated at membranes or in extracellular material. | That pattern is discordant with the reported intracellular localisation (HPA: nuclear and cytoplasmic tissue IHC; UniProt Q8N187: Nucleus, no transmembrane segment). | Check tissue morphology, no-primary and detection controls, and whether the apparent outline is background before scoring a positive (general IHC practice). |
| An unexpected cell type stains more strongly than the HPA-listed cells. | Cross-reactivity or endogenous detection activity is possible; HPA's tissue observations do not validate every cell type (general IHC practice; HPA: tissue IHC reliability). | Compare the same section's expected cell population, review controls, and report the unexpected signal as unresolved until checked (HPA: High cell-type observations; general IHC practice). |
| A Low HPA tissue shows faint staining, or an unlisted tissue shows signal. | Low means observed low staining, and HPA reports Low tissue RNA specificity; neither designation makes that tissue a negative control (HPA: Low tissue IHC observations and RNA specificity). | Record cell type, compartment, and intensity, and compare with a listed High population in the same staining run (HPA: tissue IHC observations; general IHC practice). |
| Q: Should ICC-IF show exactly the same compartment pattern as paraffin-section IHC? | HPA ICC-IF places CARF mainly in vesicles, additionally in nucleoli fibrillar centers and cytosol; its tissue IHC profile is nuclear and cytoplasmic (HPA: subcellular ICC-IF and tissue IHC). | A: Interpret each application against its own HPA observation and antibody status. This section provides no ICC-IF protocol; HPA034557 is ICC Approved, while HPA043191 has ICC approval without a listed IHC status (HPA: antibody statuses). |
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
🧪Start with appendix: its glandular cells should stain (HPA: High in appendix glandular cells). HPA detects CARF in all 45 scored tissues, so use no-primary and isotype controls in place of a negative tissue; adjacent cells without visible signal should show only background and must not be treated as proven CARF-negative (HPA: no negative tissue listed).
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: Run a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, immunoglobulin class and clonality where applicable (standard IHC practice). For a biological specificity check, use knockout material or peptide blocking; the selected antibody’s paraffin-section caption documents peptide blocking (A30570 tissue-IHC caption). Quench endogenous peroxidase and assess signal from inflammatory cells in appendix (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and its effect on CARF staining are unreported in the supplied evidence; the selected paraffin-section caption also leaves the fixative unreported (A30570 tissue-IHC caption). Antigen-retrieval dependency is unreported, so evaluate retrieval empirically for paraffin IHC (standard IHC practice). The evidence does not establish that frozen sections or IF are easier; inflammatory cells in appendix can contribute endogenous peroxidase signal that complicates chromogenic interpretation (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, cellular compartment, controls, and scoring before interpreting chromogenic signal.
How should I retrieve CARF antigen when nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Let sections cool in retrieval buffer before washing, and process comparison slides together so temperature and timing stay consistent (standard IHC practice). If staining remains weak, compare a shorter or longer heating interval on adjacent sections while monitoring tissue damage (standard IHC practice). Score nuclei separately from cytoplasm because UniProt lists a nuclear location, while tissue IHC also reports cytoplasmic expression (UniProt Q8N187; HPA: nuclear and cytoplasmic expression). Keep retrieval conditions identical across samples used for quantitative comparison (standard IHC practice).
Could fixation explain variable CARF staining between paraffin blocks?
CARF-specific fixation sensitivity is unknown: the selected paraffin-section image does not state its fixative (caption A30570). Record the fixative and fixation time for each block, then compare blocks only after matching retrieval, antibody incubation, and detection conditions (standard IHC practice). If archival blocks differ, stain adjacent sections in one run with a shared reference section and a no-primary control (standard IHC practice). Inspect morphology and staining at tissue edges, where processing differences can complicate interpretation (standard IHC practice). Do not assign a fixation effect to CARF from its reported nuclear location or absence of annotated glycosylation (UniProt Q8N187).
Should CARF staining be nuclear when cytoplasmic signal is also visible?
Evaluate nuclear and cytoplasmic staining as separate readouts: UniProt lists CARF in the nucleus, and tissue IHC reports both compartments (UniProt Q8N187; HPA: nuclear and cytoplasmic expression). Assess each compartment in intact cells with a clear counterstained nucleus, using the same scoring rules across sections (standard IHC practice). Vesicular or nucleolar patterns seen by ICC/IF can guide follow-up imaging, but their tissue-section appearance needs direct confirmation (HPA subcellular: vesicles, nucleoli fibrillar center, and cytosol). If diffuse cytoplasmic color overwhelms nuclear detail, review retrieval, antibody concentration, and detection time on adjacent sections (standard IHC practice). Record the cell type as well as the compartment (standard IHC practice).
Can an unknown antibody epitope affect how I interpret CARF staining?
CARF has 3 annotated isoforms, so establish which isoforms the antibody can recognize before assigning staining to a specific variant (UniProt Q8N187). The supplied record does not map this antibody's epitope; peptide blocking in the selected paraffin-section image supports competition by that peptide without identifying isoform coverage (caption A30570). If an epitope sequence becomes available, align it against isoforms 1, 2, and 3 and document shared and missing regions (UniProt Q8N187; standard sequence-analysis practice). Compare staining across matched sections using identical retrieval and detection settings (standard IHC practice). Do not interpret compartment differences as isoform-specific without an isoform-discriminating reagent or independent evidence (standard IHC practice).
How can IF help resolve ambiguous CARF staining in tissue?
Use IF as a separate localization experiment, multiplexing CARF with a marker for the expected cell type, such as Purkinje cells in cerebellum (HPA: high in Purkinje cells). Choose spectrally separated fluorophores and place the weaker signal in a longer-wavelength channel if tissue autofluorescence obscures it (standard IF practice). Include single-label and no-primary controls to distinguish channel bleed-through and autofluorescence from CARF signal (standard IF practice). CARF has no annotated transmembrane segment; access to nuclear, cytosolic, and vesicular targets therefore calls for a controlled permeabilization comparison rather than an assumed membrane-side epitope (UniProt Q8N187; HPA subcellular; standard IF practice). Score IF and chromogenic IHC independently (standard practice).
What should I check when CARF DAB staining looks diffuse?
First compare the stained section with a no-primary control and inspect whether color follows cells or spreads across folds, edges, and damaged areas (standard IHC practice). Quench endogenous peroxidase before chromogenic detection, then check blocking, wash stringency, and DAB development time on matched sections (standard IHC practice). The selected image shows peptide-blocked staining in paraffin-embedded human lung carcinoma tissue, but its caption does not specify fixation (caption A30570). A positive signal in that image cannot establish that every diffuse signal in another section is CARF (caption A30570; standard IHC interpretation). Reassess compartment and cell identity against the reported tissue pattern (HPA: nuclear and cytoplasmic expression).
How should I quantify CARF across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define regions and cell types before scoring, then report the percentage of positive cells and a compartment-specific H-score for nuclear and cytoplasmic staining (standard IHC scoring practice; HPA: nuclear and cytoplasmic expression). Calculate each H-score as the sum of staining-intensity categories multiplied by their respective cell percentages, using a consistent category scale (standard IHC scoring practice). Normalize positive-cell counts to all evaluable cells of the same type, or report positive-cell density per mm² of viable tissue (standard histology practice). Exclude folds and necrotic areas under a prespecified rule, and keep illumination, retrieval, and DAB development consistent (standard IHC practice). Record controls and section quality alongside scores (standard IHC practice).
How can I distinguish credible CARF staining from artefact?
Look for reproducible cellular staining with identifiable nuclei, then score nuclear and cytoplasmic signal separately (UniProt Q8N187: nucleus; HPA: nuclear and cytoplasmic expression). High staining in Purkinje cells or bronchial respiratory epithelial cells is compatible with the reported tissue profile, but cell identity alone does not prove specificity (HPA: high in those cell types; standard IHC interpretation). Treat signal limited to section edges, necrosis, or folds as suspect and compare it with a no-primary control (standard IHC practice). Check whether endogenous peroxidase contributes to DAB color, and repeat questionable regions on an adjacent section (standard IHC practice). Interpret peptide competition as supporting evidence within the selected image's context (caption A30570).
Boster reagents
Best CARF / Calcium-responsive transcription factor IHC Antibodies
A30570 has an IHC image from human lung carcinoma paraffin sections (A30570 IHC image caption). IF/ICC are listed applications, but no IF image is supplied (A30570 catalog applications and IF image list).
Real IHC data
Anti-ALS2CR8 Antibody
Cat # A30570
A30570 will render with an IHC image of paraffin-embedded human lung carcinoma tissue and a peptide-blocked comparison (A30570 IHC image caption). It lists IHC, IF and ICC applications and Human, Mouse and Rat reactivity (A30570 catalog applications and reactivity).
Which to pick: For tissue IHC, choose A30570 at 1:100–1:300 for paraffin sections; its own image shows human lung carcinoma tissue, but the fixative is unreported (A30570 IHC image caption; datasheet: 1:100–1:300). For IF/ICC, A30570 lists both applications and an IF dilution of 1:50, though no IF image is supplied (A30570 catalog applications, IF dilution and IF image list). For cross-species work, A30570 is rabbit polyclonal and lists Human, Mouse and Rat reactivity; its supplied IHC image documents human tissue only (A30570 catalog host, dilution_raw, reactivity and IHC image caption).
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 staining observed; nucleus annotated (HPA tissue IHC; 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 sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage unknown (UniProt); Regulation: CARF expression regulation unannotated (UniProt). Evidence: UniProt Q8N187, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the nucleoli…, Datasheet A30570, PMC5348329. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CARF/immunohistochemistry-ihc-guide.