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CRTC3 / CREB-regulated transcription coactivator 3 · IHC design guide
Design Immunohistochemistry for CRTC3
Plan CRTC3 staining in paraffin sections using high-staining adrenal glandular cells and undetected adipocytes as controls (HPA tissue IHC). Score nuclear and cytoplasmic staining separately, since nuclear expression predominates across tissues and activation can promote nuclear translocation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRTC3 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 | Mainly nuclear; cytoplasmic staining also occurs (HPA tissue IHC) | |
| Staining pattern | Nuclear in most tissues; cytoplasmic in several (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04568-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04568-2) | |
| Caveat | Antibody staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | Activation promotes nuclear translocation (UniProt) | |
| Isoform / epitope | 2 isoforms; confirm antibody epitope coverage (UniProt) |
Section 1
Recommended CRTC3 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 heat retrieval (datasheet: A04568-2). The published IHC reports below provide additional tissue and staining details (PMC10477178; PMC8581419).
| Sample | Paraffin-embedded human gallbladder carcinoma tissue; fixative not specified (datasheet A04568-2) |
| Fixation | Image fixative and duration unreported (datasheet A04568-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A04568-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04568-2) |
| Primary antibody | Rabbit anti-CRTC3, 2-5 μg/ml (datasheet A04568-2) |
| Primary incubation | Overnight at 4 °C (datasheet A04568-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04568-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CRTC3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in most tissues. Additional cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A04568-2); the skin IHC report specifies a retrieval solution without its composition (PMC8581419).
Section 2
What Is the Expected CRTC3 Staining Pattern?
CRTC3 should stain mainly in nuclei, with cytoplasmic staining possible (HPA: nuclear expression in most tissues; UniProt Q6UUV7: mainly nuclear, also cytoplasmic). Look for signal in the specific cells HPA rates High, including adrenal glandular cells, ovarian follicle cells and skin fibroblasts (HPA: High). CRTC3 has no transmembrane segment (UniProt Q6UUV7 topology). HPA rates its tissue IHC profile Approved but reports low consistency with RNA expression (HPA: reliability description).
| Distinct nuclear staining in adrenal glandular cells or ovarian follicle cells, with some cytoplasmic signal. | This fits the reported compartment and cell pattern (HPA: nuclear expression in most tissues, additional cytoplasmic expression in several; High in these cells). Score the named cell population and nuclear signal separately; a High HPA rating does not mean every cell on the section must stain. |
| Strong membrane rim or extracellular staining dominates, while nuclei are unstained. | This conflicts with the reported nuclear and cytoplasmic locations and the absence of a transmembrane segment (HPA: tissue IHC profile; UniProt Q6UUV7 topology). Treat the pattern as suspect and compare a control section before assigning it to CRTC3 (general IHC practice). |
| Signal appears mainly in an HPA Not detected cell population, such as adipocytes. | HPA reports CRTC3 as Not detected in adipocytes, so this pattern warrants a specificity check (HPA: adipose tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice); the HPA rating applies to adipocytes, not automatically to every cell in adipose tissue. |
| Uniform chromogen haze covers cells and surrounding tissue without clear nuclear boundaries. | The distribution is hard to reconcile with the reported nuclear emphasis (HPA: tissue IHC profile). Nonspecific primary or detection reagent binding, or endogenous detection activity, can produce background (general IHC practice); do not score the haze as positive nuclei. |
| No discernible signal appears in a section containing HPA High cells. | Check that the relevant cells are present: HPA rates salivary glandular cells, skin fibroblasts and smooth muscle cells High (HPA: tissue IHC). A blank result could reflect assay failure or a specimen difference (general IHC practice). HPA's Approved profile has low consistency with RNA expression, so one blank section is not conclusive (HPA: reliability description). |
💡Expected CRTC3 appearanceA convincing positive is clear, predominantly nuclear chromogen in the named HPA High cell population, with possible cytoplasmic signal; dominant membrane or extracellular staining is suspect (HPA: tissue IHC profile and High cell ratings; UniProt Q6UUV7 topology).
| Compartment and signaling state | CRTC3 is mainly nuclear and can also occupy the cytoplasm (UniProt Q6UUV7: subcellular location); UniProt reports nuclear translocation after adenylyl cyclase or MAP kinase activation (UniProt Q6UUV7). Interpret a nuclear-to-cytoplasmic difference in context rather than requiring one fixed ratio in every specimen. |
| Choice of positive and low-signal cells | Use the named cell population when judging a control: adrenal glandular cells and ovarian follicle cells are High, whereas adipocytes and duodenal glandular cells are Not detected (HPA: tissue IHC). A tissue name alone does not specify which cells should stain. |
| Strength of IHC validation | The tissue profile is Approved and has low consistency between antibody staining and RNA expression (HPA: reliability description). HPA063691 is IHC Approved; its Enhanced rating applies to ICC, while HPA043735 has no listed IHC rating (HPA: antibody validation). |
| Isoforms and antibody coverage | UniProt lists isoforms 1 and 3 (UniProt Q6UUV7: isoforms). The supplied record does not locate an antibody epitope or establish coverage of either isoform, so an isoform-specific explanation for weak staining remains unverified. |
| Topology and processing | CRTC3 has no transmembrane segment or signal peptide, and its annotated chain spans residues 1–619 (UniProt Q6UUV7: topology and processing). These annotations support an intracellular reading of the stain; they do not establish fixation sensitivity or an antigen retrieval setting. |
| Situation | Likely cause | Next action |
|---|---|---|
| An HPA High control shows no nuclear stain. | The expected cell population may be absent from the field, or the IHC run may have failed (HPA: cell-specific High ratings; general IHC practice). | Confirm the named cells on a counterstained section, then review the IHC-validated antibody's documented retrieval, dilution and detection conditions (general IHC practice). Interpret a persistent blank result alongside HPA's low staining–RNA consistency (HPA: reliability description). |
| Membrane-like or extracellular chromogen is stronger than nuclear signal. | That distribution conflicts with HPA's nuclear emphasis and UniProt's lack of a transmembrane segment (HPA: tissue IHC profile; UniProt Q6UUV7 topology). | Compare a named HPA High cell population and a primary-antibody omission control; review blocking and detection background before scoring (general IHC practice). |
| Adipocytes or another HPA Not detected cell population appears strongly positive. | Off-target binding or endogenous detection activity may account for unexpected staining (HPA: Not detected in adipocytes; general IHC practice). | Check a reagent control and the relevant endogenous-activity block for the detection chemistry; judge the result by the identified cell type, not the tissue label (general IHC practice). |
| The whole section has diffuse brown background. | Nonspecific binding or endogenous activity can obscure nuclear boundaries in chromogenic IHC (general IHC practice). | Review blocking, primary-antibody dilution and detection controls, then score only distinguishable cellular signal (general IHC practice; HPA: predominantly nuclear tissue profile). |
| Cytoplasmic signal is visible alongside nuclear stain. | Additional cytoplasmic tissue staining is reported, and CRTC3 can occur in the cytoplasm (HPA: tissue IHC profile; UniProt Q6UUV7: subcellular location). | Record nuclear and cytoplasmic compartments separately and compare the named positive cells; do not discard an otherwise convincing result solely for cytoplasmic signal (HPA: tissue IHC profile). |
| IF/ICC Q: What distribution should a fluorescent image show? | A: Mainly nucleoplasmic signal with additional cytosolic signal (HPA: enhanced ICC-IF subcellular localization). | Compare nuclear and cytosolic channels with the HPA subcellular pattern; keep IF/ICC protocol choices on its own guide page (HPA: ICC-IF subcellular record). |
Experiment evidence
IHC & IF images showing CRTC3 staining
A04568-2 · IHCIHC analysis of CRTC3 using anti-CRTC3 antibody (A04568-2). CRTC3 was detected in a paraffin-embedded section of human gallbladder carcinoma
A04568-2 · IHCIHC analysis of CRTC3 using anti-CRTC3 antibody (A04568-2). CRTC3 was detected in a paraffin-embedded section of human lymphoma tissue. Heat
A04568-2 · IFIF analysis of CRTC3 using anti-CRTC3 antibody (A04568-2). CRTC3 was detected in an immunocytochemical section of Hela cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140577-CRTC3/tissue/cerebral+cortex#imid_21170683
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140577-CRTC3/tissue/colon#imid_21170609
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140577-CRTC3/tissue/liver#imid_21170610
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000140577-CRTC3/tissue/kidney#imid_21170611
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA043735; green = CRTC3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA043735; green = CRTC3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRTC3 IHC & IF
🧪Run adrenal gland first and expect staining in its glandular cells (High; HPA: adrenal gland, glandular cells); use duodenal glandular cells as the tissue negative (HPA: duodenum, glandular cells, Not detected). On the adrenal slide, morphologically distinct cells that show only background chromogen can serve as internal background references, but HPA does not identify a validated negative cell type within that tissue (HPA: adrenal gland row lists glandular cells only).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CRTC3 in A-431, U-251MG, U2OS, HeLa, SiHa, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a matched nonimmune rabbit IgG isotype control (caption: rabbit anti-CRTC3; standard IHC practice); use matched CRTC3-knockout material as a biological negative if available (standard IHC practice). Quench endogenous peroxidase and assess endogenous biotin background in the adrenal section because the reported detection uses a streptavidin–biotin complex with DAB (caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A04568-2 paraffin-section caption does not state a fixative (caption: fixative unreported). The reported paraffin protocol uses heat-mediated retrieval in EDTA at pH 8.0, but the supplied evidence does not establish that retrieval is required or that frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images). In adrenal tissue, endogenous brown pigment can complicate interpretation of brown DAB signal, so assess cell morphology and the control slides together (standard histology/IHC practice).
HPA tissue IHC evidence for CRTC3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data.). 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 | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | High | Protein (IHC) | HPA → |
| Ovary | Follicle cells | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CRTC3 IHC Tips
Use compartment-aware controls and matched processing to troubleshoot CRTC3 staining in paraffin sections; assess IF on its separate guide page.
How should I adjust retrieval when CRTC3 staining is weak or uneven?
Start with heat-mediated EDTA at pH 8.0 for CRTC3 paraffin-section IHC (datasheet A04568-2). Keep heating, cooling and section thickness consistent across test and control sections, then inspect both staining and tissue morphology (standard IHC practice). Compare nuclear signal in an appropriate positive control, such as ovarian follicle cells, where staining is reported as high (HPA: High in ovarian follicle cells). If signal remains weak, optimise heating time on adjacent sections before changing buffer; document any fallback buffer separately from the tested EDTA condition (standard IHC practice).
Could fixation explain absent or patchy CRTC3 staining?
The CRTC3 paraffin-section caption does not report a fixative, so target-specific sensitivity to fixation is unknown (datasheet A04568-2: fixative not stated). Record the fixative, fixation duration, section age and processing history for each specimen before attributing a weak result to CRTC3 biology (standard IHC practice). Compare adjacent sections processed together, with a positive-control section in the same staining run, to identify variation introduced before antibody incubation (standard IHC practice). If staining differs between processing batches, optimise the documented preparation conditions and retrieval while checking morphology; do not infer a CRTC3-specific fixation effect from tissue patterns alone (standard IHC practice).
What should I make of nuclear and cytoplasmic CRTC3 staining?
Assess nuclear and cytoplasmic DAB staining separately because CRTC3 is reported in both compartments and appears mainly nuclear (UniProt Q6UUV7 subcellular location). Nuclear expression is seen in most tissues, with additional cytoplasmic expression in several tissues (HPA: tissue IHC profile). Compare compartment patterns within the same cell type and across identically processed sections before interpreting a shift as biological (standard IHC practice). Nuclear translocation can follow adenylyl cyclase or MAP kinase activation (UniProt Q6UUV7 subcellular location), but a static paraffin section cannot establish that a pathway was activated; report the observed distribution and its controls (standard IHC interpretation).
Could isoforms or epitope accessibility alter the staining pattern?
CRTC3 has two listed isoforms, 1 and 3, so establish whether the antibody's disclosed immunogen is shared before interpreting staining as total CRTC3 (UniProt Q6UUV7 isoforms). Its epitope is not specified in the supplied product caption, which prevents an isoform-specific claim from that image (datasheet A04568-2 caption). Several modified residues are annotated, including Ser-162 phosphorylation by SIK2, but no supplied evidence establishes that these modifications change this antibody's binding in sections (UniProt Q6UUV7 modified residues). If an unexpected compartment or cell population stains, compare with an independently validated epitope where available and document differences without assigning them to an isoform (standard IHC practice).
How can I check CRTC3 localisation by multiplex IF?
Plan IF as a separate assay: CRTC3 is mainly nucleoplasmic with additional cytosolic localisation in the reported subcellular images (HPA: subcellular localisation). Pair CRTC3 with a marker for the expected cell population, such as a T-lymphocyte marker when examining lymphoid cells, because lymphocytes are a reported expression site (UniProt Q6UUV7 tissue specificity). Choose a CRTC3 fluorophore channel after viewing unstained tissue for autofluorescence, and include single-label controls to check spectral bleed-through (standard IF practice). Permeabilise sufficiently to access nuclear and cytosolic CRTC3, which has no transmembrane segment, while checking whether morphology and the cell-marker signal remain intact (UniProt Q6UUV7 topology; standard IF practice).
How do I distinguish diffuse background from weak CRTC3 signal?
Compare each stained section with a no-primary control and examine whether DAB deposits follow cells or appear across stroma, edges and damaged regions (standard chromogenic IHC practice). For the catalog antibody, the documented tissue procedure used 10% goat serum blocking, 2 μg/ml primary overnight at 4°C, and a biotinylated secondary for 30 minutes at 37°C (datasheet A04568-2). Check endogenous peroxidase blocking and reagent-only controls as general DAB workflow checks, particularly if the background persists without primary antibody (standard chromogenic IHC practice). A plausible CRTC3 result should retain interpretable cellular distribution, chiefly nuclear with possible cytoplasmic signal (HPA: tissue IHC profile).
How should I quantify CRTC3 across heterogeneous paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score nuclear and cytoplasmic staining separately within a prespecified cell population, because both compartments are reported for CRTC3 (HPA: tissue IHC profile). Record the percentage of positive cells and an intensity-based H-score, or count positive cells per mm² when cell density varies across regions (standard IHC quantification practice). Normalise each readout to the number of eligible cells or evaluated tissue area, and apply one threshold and exposure-independent scoring rule to all sections (standard chromogenic IHC practice). Exclude folds, necrosis and section edges before scoring, and retain control-section results with every batch because antibody staining has low consistency with RNA expression in the tissue survey (HPA: Approved reliability description).
When is an apparent CRTC3-positive cell convincing?
Look for reproducible cellular staining with an interpretable nuclear component; CRTC3 appears mainly nuclear, although cytoplasmic localisation is also reported (UniProt Q6UUV7 subcellular location; HPA: tissue IHC profile). Check cell identity against the region being scored: high staining is reported in appendix lymphoid tissue and ovarian follicle cells, while gallbladder glandular cells are reported at low levels (HPA: tissue IHC profile). Discount signal confined to section edges, necrotic zones or no-primary controls, and investigate persistent DAB deposits as possible endogenous enzyme signal (standard chromogenic IHC practice). Treat a surprising distribution cautiously because the tissue survey reports low consistency between antibody staining and RNA expression (HPA: Approved reliability description).
Boster reagents
Best CRTC3 / CREB-regulated transcription coactivator 3 IHC Antibodies
A04568-2 has human paraffin-section IHC images and a HeLa IF image (A04568-2 image captions). M04568 lists IHC and ICC/IF applications with human, mouse, and rat reactivity (M04568 catalog).
Real IHC data
Anti-CRTC3 Antibody ®
Cat # A04568-2
A04568-2 has IHC images from human gallbladder carcinoma, lymphoma, breast cancer, and colonic adenocarcinoma paraffin sections, plus a HeLa IF image (A04568-2 image captions). M04568 lists IHC and ICC/IF applications and human, mouse, and rat reactivity; no image is supplied (M04568 catalog).
Which to pick: Choose A04568-2 for human tissue IHC because its own paraffin-section captions document EDTA retrieval and chromogenic detection (A04568-2 IHC captions). For IF/ICC, A04568-2 has a HeLa IF image; choose M04568 when mouse or rat reactivity is needed, based on its listed reactivity and applications (A04568-2 IF caption; M04568 catalog). The A04568-2 IHC captions do not report a fixative, so fixation is unconfirmed (A04568-2 IHC captions). The selected A04568-2 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A04568-2).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6UUV7 (CRTC3_HUMAN, CREB-regulated transcription coactivator 3).
- Human Protein Atlas. CRTC3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CRTC3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. CRTC3 antibody validation summary (2 antibodies).
- In vitro CRISPR screening uncovers CRTC3 as a regulator of IFN-γ-induced ferroptosis of hepatocellular carcinoma. Cell death discovery 2023 — PMC10477178.
- CRTC3, a sensor and key regulator for melanogenesis, as a tunable therapeutic target for pigmentary disorders. Theranostics 2021 — PMC8581419.
- Hidradenoma in Axillary Lymph Nodes: A Diagnostic Pitfall Mimicking Metastatic Carcinoma. The American journal of surgical pathology 2026 — PMC13361949.
- PubMed PMID:14506290 — UniProt-cited evidence.
- PubMed PMID:12693554 — UniProt-cited evidence.
- PubMed PMID:16572171 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRTC3 (CREB-regulated transcription coactivator 3). Expected localisation Mainly nuclear; cytoplasmic staining also occurs (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04568-2). Validated IHC antibody: A04568-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gallbladder carcinoma tissue; fixative not specified (datasheet A04568-2); 2. Fixation: Image fixative and duration unreported (datasheet A04568-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A04568-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04568-2); 8. Primary antibody: Rabbit anti-CRTC3, 2-5 μg/ml (datasheet A04568-2); 9. Primary incubation: Overnight at 4 °C (datasheet A04568-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04568-2); Caveat: Antibody staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; confirm antibody epitope coverage (UniProt); Regulation: Activation promotes nuclear translocation (UniProt). Evidence: UniProt Q6UUV7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A04568-2, PMC10477178, PMC8581419. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRTC3/immunohistochemistry-ihc-guide.