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CLOCK / Circadian locomoter output cycles protein kaput · IHC design guide
Design Immunohistochemistry for CLOCK
Plan paraffin CLOCK IHC around widespread nuclear staining, with high signal in Leydig cells (HPA tissue IHC). Start the catalog antibody at 2–5 μg/mL (datasheet: RP1082), and account for circadian changes in localisation when interpreting staining (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CLOCK 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 in tissue (HPA tissue IHC); cytoplasm possible (UniProt) | |
| Staining pattern | Widespread nuclear staining; high in Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis+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 | Circadian phase may shift nuclear versus cytoplasmic signal (UniProt) | |
| Regulation | Circadian nuclear shuttling (UniProt) | |
| Isoform / epitope | No annotated isoforms; one intracellular 1–846 chain (UniProt) |
Section 1
Recommended CLOCK IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with one published liver IHC protocol for CLOCK (PMC5985434).
| Sample | Paraffin-embedded human thyroid carcinoma tissue; fixative not specified (datasheet M00064) |
| Fixation | Image fixative and duration unreported (datasheet M00064); 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 monoclonal (clone ACCH-3) anti-CLOCK, 1:50 (datasheet M00064) |
| 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 | CLOCK-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear 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); compare citrate pH 6.0 if needed (PMC5985434).
Section 2
What Is the Expected CLOCK Staining Pattern?
CLOCK is predominantly nuclear in tissue IHC (HPA: ubiquitous nuclear expression; Supported reliability, with medium consistency against RNA data). Expect the clearest signal in testicular Leydig cells, where staining is High; several other listed cell populations show Medium staining (HPA: tissue IHC). Some cytoplasmic signal can be biologically plausible because CLOCK moves between nucleus and cytoplasm under circadian and BMAL1-dependent regulation (UniProt O15516: subcellular location). CLOCK has no transmembrane segment (UniProt O15516: topology).
| Distinct nuclear staining in Leydig cells; weaker nuclear staining across other tissues. | This fits the reported pattern: Leydig cells are High, while adipocytes and several glandular or neuronal cell populations are Medium (HPA: tissue IHC). Compare compartments and cell populations as well as intensity; the tissue profile is ubiquitous and has Supported reliability with medium RNA–staining consistency (HPA: tissue IHC). |
| Predominantly cytoplasmic staining, with little nuclear signal. | Review the slide before calling this artefact. CLOCK can shuttle between cytoplasm and nucleus; its phosphorylated form is nuclear, while its nonphosphorylated form is reported only in cytoplasm (UniProt O15516: subcellular location). A predominantly cytoplasmic tissue pattern differs from HPA’s ubiquitous nuclear IHC profile and merits antibody and detection controls (HPA: tissue IHC; general IHC practice). |
| Strong staining appears chiefly in an unexpected cell population. | Compare the actual cell identity with the HPA tissue profile before assigning specificity: Leydig cells are High, while several other listed populations are Medium or Low (HPA: tissue IHC). Unexpected staining could reflect antibody cross-reactivity or endogenous chromogenic activity; inspect controls and tissue morphology before interpreting it as CLOCK (general IHC practice). |
| Diffuse chromogen covers nuclei, cytoplasm, and tissue background. | A diffuse pattern cannot establish CLOCK localization against the reported nuclear tissue profile (HPA: tissue IHC). Excess primary antibody, incomplete blocking, endogenous detection activity, or nonspecific secondary binding are general IHC possibilities; a primary-omission control helps distinguish detection background from primary-dependent staining (general IHC practice). |
| No detectable staining in testicular Leydig cells. | This conflicts with the High Leydig-cell staining reported by HPA, but one negative section does not establish absence of CLOCK (HPA: tissue IHC). Check that Leydig cells are present, then review the antibody’s IHC validation, retrieval and detection conditions alongside an appropriate positive control (HPA: antibody validation; general IHC practice). |
💡Expected CLOCK appearanceCall a section positive when staining is chiefly nuclear in identifiable cells, with High signal in Leydig cells and potentially Medium signal in listed populations; diffuse background or strong staining confined to unexpected cells needs control-based review (HPA: tissue IHC; general IHC practice).
| Circadian compartment changes | Nuclear-to-cytoplasmic distribution can vary with circadian regulation and BMAL1 dependence; record collection time when comparing experiments (UniProt O15516: subcellular location; general study-design practice). |
| Choice of reference tissue | Leydig cells provide a High-staining reference; small-intestinal glandular cells, cholangiocytes, and pancreatic exocrine cells are Low, not established negatives (HPA: tissue IHC). |
| Antibody evidence | Two listed rabbit polyclonal antibodies have Supported IHC status; HPA gives the tissue profile Supported reliability with medium RNA–staining consistency (HPA: antibody validation and tissue IHC). |
| Molecular location and processing | CLOCK has no transmembrane segment, signal peptide, or propeptide; the supplied record describes one chain spanning residues 1–846 (UniProt O15516: topology and processing). |
| Situation | Likely cause | Next action |
|---|---|---|
| Leydig-cell nuclei are blank. | The result disagrees with the High Leydig-cell reference; a failed IHC workflow or absence of identifiable Leydig cells remains possible (HPA: tissue IHC; general IHC practice). | Confirm tissue morphology and run a known-positive section; review the catalog antibody’s IHC-P instructions, including its specified retrieval and dilution, before adjusting the workflow (general IHC practice). |
| Only weak staining appears in a Low-listed population. | Low staining is reported for several populations, including small-intestinal glandular cells and cholangiocytes; these are poor sole checks for assay performance (HPA: tissue IHC). | Include a section with identifiable Leydig cells as the stronger reference and compare nuclear localization under the same staining run (HPA: High in Leydig cells; general IHC practice). |
| Chromogen is widespread or obscures cell boundaries. | Nonspecific antibody binding or endogenous detection activity can obscure a compartment-specific readout (general IHC practice). | Inspect a primary-omission control and review blocking, primary concentration, washes, and endogenous-enzyme controls as appropriate to the chromogenic system (general IHC practice). |
| The strongest signal is cytoplasmic. | Cytoplasmic CLOCK is plausible, but a chiefly cytoplasmic slide diverges from HPA’s ubiquitous nuclear tissue pattern (UniProt O15516: subcellular location; HPA: tissue IHC). | Check nuclear counterstain and morphology, compare control sections and collection times, and avoid scoring cytoplasmic intensity alone as confirmation of specific staining (general IHC practice; UniProt O15516: circadian shuttling). |
| A different cell population stains strongly. | CLOCK expression is broad, but the observed population may differ from the listed HPA pattern; cross-reactivity or endogenous chromogenic activity also remains possible (HPA: tissue IHC; general IHC practice). | Identify the cells morphologically, compare their reported HPA level, and inspect primary-omission and other appropriate assay controls before assigning the signal to CLOCK (HPA: tissue IHC; general IHC practice). |
| What pattern should an IF/ICC image show? | The HPA ICC-IF summary places CLOCK mainly in the nucleoplasm and additionally in vesicles; this is a separate imaging context from paraffin-section IHC (HPA: subcellular ICC-IF). | Interpret an IF/ICC image against its own nuclear and vesicular reference pattern; do not use it to override the predominantly nuclear tissue-IHC profile (HPA: subcellular ICC-IF and tissue IHC). |
Experiment evidence
IHC & IF images showing CLOCK staining
M00064 · IHCImmunohistochemical analysis of paraffin-embedded human thyroid carcinoma, using CLOCK Antibody.
M00064 · IFImmunofluorescent analysis of HeLa cells, using CLOCK Antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000134852-CLOCK/tissue/cerebral+cortex#imid_6902192
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000134852-CLOCK/tissue/colon#imid_926974
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000134852-CLOCK/tissue/liver#imid_6902111
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000134852-CLOCK/tissue/kidney#imid_926976
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001867; green = CLOCK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001867; green = CLOCK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CLOCK IHC & IF
🧪Run testis first and require staining in Leydig cells (HPA: High in Leydig cells). HPA detects CLOCK in all 45 scored tissues, so use no-primary and isotype controls for the negative; any unstained neighboring cells are a background reference, not a validated CLOCK-negative population (HPA: no negative tissue rows).
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: None in HPA: CLOCK 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 CLOCK in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a matched isotype control with the same host species and antibody class; use CLOCK knockout tissue or a peptide block, if available, to test target specificity (standard IHC practice). In testis, check endogenous peroxidase and, if using a biotin-based detection system, endogenous biotin; assess autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M00064 paraffin-section caption does not state a fixative (M00064 caption). Retrieval dependency is unreported, and the supplied evidence does not establish whether frozen sections or IF are easier; nucleoplasmic IF signal has been observed (HPA: supported nucleoplasm localization). No testis-specific artifact is reported in the supplied evidence; use the no-primary testis section to assess background (standard IHC practice).
HPA tissue IHC evidence for CLOCK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CLOCK is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CLOCK IHC Tips
Troubleshoot CLOCK staining in paraffin sections by checking retrieval, compartment, controls and sampling time before comparing chromogenic signal across specimens.
How should I retrieve CLOCK antigen when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min in paraffin sections (page retrieval rule: nuclear antigen). Let sections cool in the retrieval buffer, then keep section thickness, heating and chromogenic development consistent across the comparison (standard IHC practice). If staining remains weak, test a different retrieval buffer or heating duration on adjacent sections while holding antibody concentration constant (standard IHC practice). Compare nuclear signal with background in a no-primary control and assess tissue preservation before accepting a stronger condition (standard IHC practice). The selected image establishes staining in paraffin-embedded human thyroid carcinoma but reports no fixative (M00064 caption).
Could fixation explain variable CLOCK staining between paraffin blocks?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected caption identifies paraffin-embedded human thyroid carcinoma but gives no fixative (M00064 caption). Record each block’s fixative, fixation interval and processing history before comparing CLOCK staining (standard IHC practice). For prospective specimens, process matched pieces under the same documented conditions and stain them together using the page retrieval condition, Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval rule; standard IHC practice). If one block stains poorly, assess its morphology and another control antigen before attributing the difference to CLOCK biology (standard IHC practice). Neither the tissue staining profile nor CLOCK’s phosphorylation sites establishes a fixation effect (HPA tissue IHC; UniProt O15516).
Should CLOCK staining be nuclear or cytoplasmic in tissue sections?
Score nuclear and cytoplasmic staining separately: CLOCK is reported in both compartments, with circadian and BMAL1-dependent shuttling (UniProt O15516: subcellular location). A predominantly nuclear pattern has independent support from tissue staining and a supported nucleoplasmic location in cell imaging (HPA tissue IHC; HPA subcellular). CLOCK’s phosphorylated form is reported in the nucleus, while its nonphosphorylated form is reported only in the cytoplasm (UniProt O15516: subcellular location). Record sampling time and preserve identical counterstain and detection settings when comparing compartments (UniProt O15516: circadian regulation; standard IHC practice). Treat isolated diffuse cytoplasmic color cautiously until it reproduces in intact cells and clears in a no-primary control (standard IHC practice).
How should an unknown epitope affect CLOCK antibody validation?
The supplied record lists 0 isoforms, but it does not identify the catalog antibody’s epitope (UniProt O15516; supplied product evidence). CLOCK contains a bHLH region at residues 34–84, two PAS regions and a PAC region, so an undisclosed immunogen cannot be assigned to any one domain (UniProt O15516: domains). Several phosphorylation sites are reported, including serines 38, 42 and 427; their effect on this antibody’s staining is unknown (UniProt O15516: modified residues; supplied product evidence). Request the immunogen or epitope information before interpreting loss of staining as loss of CLOCK protein (standard antibody validation practice). Compare independent epitope reagents or a validated perturbation using matched paraffin sections if the conclusion depends on specificity (standard IHC practice).
How can I check CLOCK localisation by multiplex immunofluorescence?
Design IF as a separate validation experiment: the selected catalog image documents paraffin-section chromogenic IHC, with no IF protocol supplied here (M00064 caption; supplied product evidence). Pair CLOCK with a marker validated for the expected cell population and use a nuclear counterstain to judge compartment overlap (standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with less tissue autofluorescence, checking single-stain and unstained controls (standard IF practice). CLOCK has no transmembrane segment and is reported in the nucleus and cytoplasm, so assess nuclear access with a titrated permeabilisation step appropriate to the specimen (UniProt O15516: topology and subcellular location; standard IF practice). Confirm that the chosen fixation and permeabilisation preserve both markers, since target-specific IF fixation sensitivity is not supplied (supplied product evidence; standard IF practice).
How do I reduce diffuse brown staining without losing nuclear CLOCK?
Inspect a no-primary slide and the chromogen-only reaction to separate antibody-related color from endogenous enzyme activity (standard chromogenic IHC practice). Use an appropriate peroxidase block before a peroxidase-based detection system, then titrate primary antibody and shorten chromogen development if background remains high (standard IHC practice). Block nonspecific binding and wash consistently, keeping the same detection settings across comparison slides (standard IHC practice). Judge each change against intact nuclear staining because tissue IHC shows a broadly nuclear CLOCK pattern (HPA tissue IHC: ubiquitous nuclear expression). Exclude folds, damaged edges and necrotic areas from the assessment, and verify that reduced background does not erase the expected cellular pattern (standard IHC practice; HPA tissue IHC).
What is a defensible way to quantify CLOCK IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and eligible intact cells before scoring, then report nuclear CLOCK as percent-positive cells and an H-score using prespecified intensity categories (standard IHC quantification practice). If spatial distribution matters, report positive-cell density per mm² of viable, annotated tissue alongside the cell-based score (standard IHC quantification practice). Normalize counts to eligible nuclei or viable area, and use identical thresholds, counterstain and image acquisition settings across slides (standard IHC quantification practice). Record collection time because CLOCK localisation is under circadian regulation (UniProt O15516: subcellular location). Keep cytoplasmic staining as a separate measure and compare like cell populations, since tissue IHC reports widespread nuclear expression (UniProt O15516: subcellular location; HPA tissue IHC).
When is a CLOCK-positive cell convincing rather than an artefact?
A convincing result has cellular staining in preserved tissue, with a nuclear component consistent with the reported tissue pattern and nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Leydig cells offer a reported high-staining reference, while several other listed cell populations show medium staining (HPA tissue IHC: positive-cell profile). Check whether signal follows damaged edges, folds or necrotic debris, and whether it persists in a no-primary or peroxidase-control slide (standard IHC practice). Cytoplasmic staining alone is not definitive artefact because CLOCK can shuttle between cytoplasm and nucleus (UniProt O15516: subcellular location). Interpret differences alongside sampling time, cell identity and controls; a single chromogenic section cannot establish a circadian rhythm (UniProt O15516: circadian regulation; standard IHC interpretation practice).
Boster reagents
Best CLOCK / Circadian locomoter output cycles protein kaput IHC Antibodies
Anti-CLOCK IHC images cover paraffin-embedded human thyroid carcinoma, human cancer tissue, and mouse and rat brain (M00064 and RP1082 image captions); IF images cover HeLa and U2OS cells (M00064 and RP1082 image captions).
Real IHC data
Anti-CLOCK Monoclonal Antibody
Cat # M00064
M00064 has an IHC image from paraffin-embedded human thyroid carcinoma and an IF image from HeLa cells; its listed reactivity is Human, Mouse, and Rat (M00064 image captions; catalog reactivity). RP1082 has IHC images from paraffin-embedded human cancer tissue, mouse brain, and rat brain, plus an IF image from U2OS cells (RP1082 image captions).
Which to pick: For tissue IHC, choose RP1082 if its documented paraffin-section workflow is useful: its captions specify EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody, but do not report the fixative (RP1082 IHC image captions). For IF/ICC, M00064 offers a rabbit monoclonal with a HeLa IF image; its IHC caption shows a paraffin-embedded section but does not report the fixative (M00064 catalog clone and applications; M00064 image captions). For cross-species planning, RP1082 lists Human, Monkey, Mouse, and Rat reactivity, while its IHC images document human, mouse, and rat tissues (RP1082 catalog reactivity; RP1082 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15516 (CLOCK_HUMAN, Circadian locomoter output cycles protein kaput).
- Human Protein Atlas. CLOCK tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CLOCK subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. CLOCK antibody validation summary (2 antibodies).
- Immunohistochemistry of the circadian clock in mouse and human vascular tissues. Vessel plus 2018 — PMC6085090.
- Differential Regulation of BMAL1, CLOCK, and Endothelial Signaling in the Aortic Arch and Ligated Common Carotid Artery. Journal of vascular research 2016 — PMC5765856.
- A comprehensive analysis of the circRNA-miRNA-mRNA network in osteocyte-like cell associated with Mycobacterium leprae infection. PLoS neglected tropical diseases 2022 — PMC9098081.
- Melatonin Attenuates Dysregulation of the Circadian Clock Pathway in Mice With CCl(4)-Induced Fibrosis and Human Hepatic Stellate Cells. Frontiers in pharmacology 2018 — PMC5985434.
- PubMed PMID:10198158 — UniProt-cited evidence.
- PubMed PMID:9205841 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CLOCK (Circadian locomoter output cycles protein kaput). Expected localisation Nuclear in tissue (HPA tissue IHC); cytoplasm possible (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00064 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human thyroid carcinoma tissue; fixative not specified (datasheet M00064); 2. Fixation: Image fixative and duration unreported (datasheet M00064); 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 monoclonal (clone ACCH-3) anti-CLOCK, 1:50 (datasheet M00064); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Circadian phase may shift nuclear versus cytoplasmic signal (UniProt); Isoform / epitope: No annotated isoforms; one intracellular 1–846 chain (UniProt); Regulation: Circadian nuclear shuttling (UniProt). Evidence: UniProt O15516, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet M00064, PMC5985434. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CLOCK/immunohistochemistry-ihc-guide.