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CRTC3 / CREB-regulated transcription coactivator 3 · IHC design guide
Design Immunohistochemistry for CRTC3
Plan chromogenic IHC on paraffin sections using the documented CRTC3 tissue pattern and cell-specific controls (HPA tissue IHC). The guide covers fixation, retrieval, detection and scoring, with nuclear staining as the principal readout (HPA tissue IHC).
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 | Predominantly nuclear; cytoplasmic in some tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in most tissues; some cytoplasmic staining (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 | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has low consistency with RNA data (HPA tissue IHC) | |
| Regulation | Nuclear shift after AC/MAPK activation (UniProt) | |
| Isoform / epitope | Two isoforms (1, 3); epitope coverage unknown (UniProt) |
Section 1
Recommended CRTC3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published CRTC3 staining in mouse HCC tissue and a published paraffin-section skin IHC workflow (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 excerpt names a retrieval solution but gives no pH (PMC8581419).
Section 2
What Is the Expected CRTC3 Staining Pattern?
CRTC3 is mainly nuclear, with cytoplasmic staining in some tissues (HPA: tissue IHC; UniProt Q6UUV7: nucleus and cytoplasm). Look for staining in the specific cell populations reported as high, such as adrenal glandular cells, ovarian follicle cells, and appendix lymphoid tissue (HPA: High). HPA rates its tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression; interpret tissue comparisons with that limitation (HPA: tissue IHC).
| Predominantly nuclear staining in adrenal glandular cells, ovarian follicle cells, or appendix lymphoid tissue. | This matches the reported compartment and high-staining cell populations (HPA: nuclear expression in most tissues; HPA: High in these cells). Compare cells within the section as well as across control sections (general IHC practice). |
| Nuclear staining accompanied by some cytoplasmic staining. | This can fit CRTC3: HPA reports additional cytoplasmic expression in several tissues, and UniProt lists both compartments (HPA: tissue IHC; UniProt Q6UUV7: subcellular location). Judge the cell population and control pattern before calling it nonspecific. |
| Predominantly membrane staining or staining confined to an unexpected compartment. | Treat this as suspect: CRTC3 has no annotated transmembrane segment and is reported in the nucleus and cytoplasm (UniProt Q6UUV7: topology and subcellular location). Check morphology, controls, and detection background before interpreting it as CRTC3. |
| Strong staining in an HPA-listed unstained cell population, such as adipocytes or colon endothelial cells. | Consider cross-reactivity or endogenous chromogen activity, then test controls (HPA: Not detected in those specific cells; general IHC practice). The HPA entries describe cell populations, not every cell in adipose tissue or colon. |
| Diffuse staining across cells and extracellular spaces, or no staining in a reported high-staining control. | Diffuse color without a discernible cellular pattern suggests background (general IHC practice). An absent control signal makes a negative test section inconclusive; HPA’s Approved profile also has low staining–RNA consistency (HPA: tissue IHC). |
💡Expected CRTC3 appearanceCall a section positive when the relevant cells show clear, predominantly nuclear CRTC3 staining, with possible cytoplasmic staining and a strong signal in reported High populations; diffuse extracellular color or isolated membrane staining is suspect (HPA: tissue IHC; UniProt Q6UUV7: topology and location; general IHC practice).
| Cell population selected for interpretation | HPA reports High staining in adrenal glandular cells, ovarian follicle cells, and appendix lymphoid tissue, but Not detected in specified populations including adipocytes and colon endothelial cells (HPA: tissue IHC). Score the named cells rather than assigning one result to an entire organ. |
| Cellular compartment and activation state | CRTC3 appears mainly nuclear and can move to the nucleus after adenylyl cyclase or MAP kinase activation (UniProt Q6UUV7: subcellular location). Cytoplasmic staining alone needs context; HPA also reports additional cytoplasmic expression in several tissues (HPA: tissue IHC). |
| Strength of the IHC evidence | The tissue profile is Approved, with low consistency between antibody staining and RNA expression (HPA: tissue IHC). HPA lists IHC as Approved for HPA063691 and gives no IHC status for HPA043735 (HPA: antibody validation); these entries do not establish equivalent IHC performance. |
| Isoforms and phosphorylation | UniProt lists isoforms 1 and 3 and multiple phosphorylated residues, including Ser-162 (UniProt Q6UUV7: isoforms and modified residues). Without an epitope map, their effects on recognition by a given IHC antibody cannot be predicted from this record. |
| Chromogenic detection background | Endogenous enzyme activity or nonspecific detection reagents can add color in chromogenic IHC (general IHC practice). Use appropriate reagent and no-primary controls when color does not follow the expected cellular pattern (general IHC practice). |
| IF/ICC Q&A: where should CRTC3 appear? | Mainly in the nucleoplasm, with additional cytosol signal (HPA: enhanced ICC-IF localization). This is localization evidence for IF/ICC; the IHC tissue pattern and its Approved rating come from HPA tissue IHC (HPA: subcellular and tissue records). |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported High cells show no chromogenic signal. | The run may have missed the target or the chosen section may differ from HPA’s observed pattern; HPA reports low staining–RNA consistency (HPA: tissue IHC; general IHC practice). | Check a reported High cell population on the same run, verify reagent and detection steps, and assess section morphology before calling the test tissue negative (HPA: High cell entries; general IHC practice). |
| Signal looks mostly membranous. | Membrane confinement conflicts with the reported nuclear and cytoplasmic locations and lack of a transmembrane segment (UniProt Q6UUV7: location and topology). | Recheck cellular boundaries and counterstain; compare with a no-primary control and a reported High control population (general IHC practice; HPA: High cell entries). |
| Uniform brown color obscures nuclei and cell boundaries. | Nonspecific detection or endogenous enzyme activity can obscure cellular staining in chromogenic IHC (general IHC practice). | Inspect reagent controls, review blocking and washing, and adjust detection conditions until the cell pattern can be read (general IHC practice). |
| Adipocytes or colon endothelial cells stain strongly. | Those specific populations are listed as Not detected by HPA; cross-reactivity or detection background is possible (HPA: tissue IHC; general IHC practice). | Confirm which cells carry the color, check a no-primary control, and compare with a reported High population before assigning target positivity (general IHC practice; HPA: High cell entries). |
| A section shows both nuclear and cytoplasmic color. | Both compartments are reported for CRTC3; HPA describes nuclear staining in most tissues with additional cytoplasmic staining in several (HPA: tissue IHC; UniProt Q6UUV7: location). | Score nuclear and cytoplasmic patterns separately in the named cells, then compare their distribution with controls; avoid treating all cytoplasmic color as artefact (general IHC practice; HPA: tissue IHC). |
| Staining changes across sections after retrieval adjustments. | Retrieval conditions can change staining in paraffin IHC, but target-specific fixation sensitivity or an optimal CRTC3 retrieval condition is unreported here (general IHC practice; supplied UniProt and HPA records). | Keep section preparation and detection consistent while comparing retrieval conditions against the same reported High control cells and background controls (general IHC practice; HPA: High cell entries). |
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; its glandular cells should stain for CRTC3 (HPA: High in adrenal gland glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, assess background in adjacent cells without specific staining, without assuming every other cell type is CRTC3-negative.
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, concentration-matched rabbit IgG isotype control, and CRTC3 knockout tissue or a peptide-block control if the immunizing peptide is available (caption: rabbit anti-CRTC3 antibody). For the caption’s biotin-based DAB detection, check endogenous peroxidase and biotin signal in the adrenal section (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A04568-2 tissue-IHC caption does not state the fixative (caption: fixative not stated). The paraffin-section example uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; the evidence also does not establish whether frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images). Assess adrenal background from endogenous peroxidase and biotin with the stated detection system (caption: SABC and DAB).
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
Troubleshoot CRTC3 staining in paraffin sections by checking retrieval, nuclear and cytoplasmic patterns, cell identity, and controls before interpreting signal.
What retrieval should I start with for CRTC3 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04568-2). The selected tissue-IHC example used that retrieval before 10% goat serum blocking and 2 μg/ml antibody overnight at 4°C (datasheet A04568-2). If staining is weak, compare a second retrieval condition on adjacent sections while holding antibody and detection conditions constant (standard IHC practice). Watch for tissue damage or diffuse background as retrieval increases, and compare nuclear signal in matched cell populations (standard IHC practice; HPA tissue IHC: nuclear expression in most tissues).
Could fixation explain weak or uneven CRTC3 staining?
CRTC3-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet A04568-2). Record the fixative, fixation duration, processing history, and section age for each specimen before comparing staining intensity (standard IHC practice). If sections differ in these variables, stain adjacent sections in the same run with the documented EDTA pH 8.0 retrieval and 2 μg/ml antibody conditions (datasheet A04568-2; standard IHC practice). Interpret a loss of nuclear staining cautiously until tissue preservation and a matched positive control are checked (HPA tissue IHC: nuclear expression in most tissues; standard IHC practice).
How should I assess nuclear versus cytoplasmic CRTC3 staining?
Score nuclear and cytoplasmic staining separately because CRTC3 is reported in both compartments and appears mainly nuclear (UniProt Q6UUV7: subcellular location). Tissue IHC shows nuclear expression in most tissues with additional cytoplasmic expression in several tissues (HPA tissue IHC). Adenylyl cyclase or MAP kinase activation can promote nuclear translocation, so a changed nuclear fraction may be biologically plausible (UniProt Q6UUV7: subcellular location). Use the counterstain to define nuclei, compare matched cell populations, and record nuclear-positive percentage alongside cytoplasmic intensity (standard IHC practice). Avoid calling diffuse chromogen outside intact cells a localisation shift without reviewing morphology and detection controls (standard IHC practice).
Could epitope choice change the CRTC3 staining pattern?
CRTC3 has 2 listed isoforms, 1 and 3, but the supplied material does not map the catalog antibody epitope to either isoform (UniProt Q6UUV7: isoforms; datasheet A04568-2). The protein has no annotated transmembrane segment and has multiple phosphorylation sites, including Ser-162 (UniProt Q6UUV7: topology and modified residues). Do not infer isoform specificity or phosphorylation sensitivity from staining alone without an epitope map and suitable controls (standard IHC practice). If sections disagree, compare the same antibody conditions across matched specimens and document compartment-specific staining before assigning the difference to an isoform or modification (standard IHC practice).
How can I check a CRTC3 pattern by multiplex immunofluorescence?
For a separate IF assay, pair CRTC3 with a marker identifying the expected cell population; lymphocytes are supported by the expression record, but marker selection depends on the specimen (UniProt Q6UUV7: tissue specificity; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning a CRTC3 channel (standard IF practice). CRTC3 is intracellular, with nuclear and cytosolic localisation and no transmembrane segment, so permeabilisation must permit access to the antibody's mapped epitope and the nucleus when nuclear staining is assessed (UniProt Q6UUV7: topology and subcellular location; standard IF practice). Keep secondary-only and single-channel controls to identify bleed-through or nonspecific fluorescence (standard IF practice).
What should I change when CRTC3 chromogen is diffuse?
First compare the primary-antibody slide with a no-primary control to identify signal from the secondary and detection chemistry (standard IHC practice). The selected paraffin-section example used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a biotin-based DAB detection system (datasheet A04568-2). Check peroxidase blocking and, with biotin-based detection, investigate endogenous biotin where background persists (standard IHC practice). Review washes, section edges, damaged areas, and DAB development before changing antibody concentration (standard IHC practice). Judge improvement by clearer staining in intact nuclei relative to surrounding tissue and the no-primary control (HPA tissue IHC: nuclear expression in most tissues; standard IHC practice).
How should I quantify CRTC3 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and regions of interest before scoring, then apply the same chromogen threshold and imaging settings across slides (standard IHC practice). Report the percentage of positive nuclei and a nuclear H-score from 0–300; score cytoplasmic intensity separately when present (standard IHC practice; HPA tissue IHC: nuclear expression with additional cytoplasmic expression). Normalise counts to the number of eligible, intact cells in each region, or report positive-cell density per mm² of viable tissue (standard IHC practice). Include matched control tissue and batch information because the HPA tissue-IHC profile reports low consistency between antibody staining and RNA expression (HPA tissue IHC: reliability description).
When is a CRTC3-positive IHC result convincing?
A convincing result follows intact cell boundaries and shows a plausible nuclear signal, with cytoplasmic staining assessed separately (UniProt Q6UUV7: subcellular location; HPA tissue IHC; standard IHC practice). Check that stained cells match the annotated population: HPA reports high staining in appendix lymphoid tissue but no detection in adipocytes (HPA tissue IHC: appendix and adipose tissue). Edge-restricted staining, necrotic areas, and signal reproduced by a no-primary control warrant review for processing or detection artefacts (standard IHC practice). Confirm the pattern on adjacent sections with the same EDTA pH 8.0 retrieval and interpret discordance cautiously because tissue-IHC staining has low consistency with RNA expression (datasheet A04568-2; HPA tissue IHC: reliability description; standard IHC practice).
Boster reagents
Best CRTC3 / CREB-regulated transcription coactivator 3 IHC Antibodies
A04568-2 has IHC images from human paraffin sections and an IF image from HeLa cells (catalog image captions); M04568 lists IHC and IF applications with human, mouse, and rat reactivity (catalog applications/reactivity).
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 an IF image from HeLa cells (catalog image captions). M04568 lists IHC and ICC/IF applications and human, mouse, and rat reactivity, but has no IHC or IF image captions in the payload (catalog applications/reactivity/image captions).
Which to pick: For human paraffin-section IHC, choose A04568-2: its own captions document staining in four tissues, including an example at 2 μg/ml after EDTA pH 8.0 retrieval; the fixative is unreported (A04568-2 IHC image captions). For IF/ICC, A04568-2 has a HeLa-cell IF image at 5 μg/ml, while M04568 lists ICC/IF without an image caption (catalog IF image captions/applications). For mouse or rat samples, M04568 is the listed option because its reactivity includes both species; it is a rabbit monoclonal, whereas A04568-2 lists human and monkey reactivity (catalog host/clone/reactivity).
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 Predominantly nuclear; cytoplasmic in some tissues (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: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA data (HPA tissue IHC); Isoform / epitope: Two isoforms (1, 3); epitope coverage unknown (UniProt); Regulation: Nuclear shift after AC/MAPK activation (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.