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CRY2 / Cryptochrome-2 · IHC design guide
Design Immunohistochemistry for CRY2
Plan paraffin CRY2 IHC with the catalog antibody at 0.5–1 μg/mL (datasheet PB9576). Assess granular cytoplasmic staining across tissues (HPA tissue IHC), and record collection time for intensity comparisons (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRY2 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 | Granular cytoplasm (HPA tissue IHC); nucleus possible (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9576) | |
| Positive control | Adrenal gland+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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9576) | |
| Caveat | Sampling time may affect intensity comparisons (UniProt) | |
| Regulation | Circadian feedback regulates CRY2 (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended CRY2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9576) is accompanied by 3 published CRY2 IHC protocols (PMC5652816; PMC4458447; PMC7476803).
| Sample | Paraffin-embedded rat liver tissue; fixative not specified (datasheet PB9576) |
| Fixation | Image fixative and duration unreported (datasheet PB9576); 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 PB9576); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9576) |
| Primary antibody | Rabbit anti-CRY2, 0.5-1μg/ml (datasheet PB9576) |
| Primary incubation | Overnight at 4 °C (datasheet PB9576) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9576) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CRY2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in a majority of tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: PB9576); citrate at pH 6.0 is a published alternative (PMC4458447).
Section 2
What Is the Expected CRY2 Staining Pattern?
CRY2 is a soluble protein found in the cytoplasm and nucleus, with no transmembrane segment (UniProt Q49AN0: subcellular location and topology). In tissue IHC, expect granular cytoplasmic staining across many cell types rather than a tissue restricted pattern (HPA tissue IHC: Supported; granular cytoplasmic expression in a majority of tissues). HPA reports High staining in several specific cell populations (HPA tissue IHC).
| Granular cytoplasmic staining appears in cells from several tissues. | This matches the predominant tissue IHC pattern (HPA tissue IHC: granular cytoplasmic expression in a majority of tissues; Supported). Assess staining in identifiable cells against adjacent background. HPA reports High staining in adrenal glandular cells, bronchial respiratory epithelial cells and bone marrow hematopoietic cells, among others (HPA tissue IHC). |
| Staining is confined to an unexpected compartment, with no convincing cytoplasmic signal. | Recheck the result before assigning it to CRY2. Nuclear staining alone is not inherently wrong because CRY2 also localizes to the nucleus (UniProt Q49AN0: subcellular location). The predominant tissue IHC observation is granular cytoplasm (HPA tissue IHC); HPA nuclear speckles come from ICC-IF, a different preparation (HPA subcellular: nuclear speckles). |
| Strong staining appears only in a cell population outside the reported positive populations. | Consider cross-reactivity or endogenous detection activity, especially if the reported granular cytoplasmic pattern is absent (HPA tissue IHC: profile). The supplied HPA list gives positive examples, not an exhaustive cell map or a validated negative population (HPA tissue IHC: positive list; negative list empty). Check morphology and appropriate staining controls before interpreting the unexpected cells. |
| Diffuse colour covers cells and surrounding tissue without a clear granular pattern. | Treat the result as background until controls and microscopy support a cellular signal. Diffuse staining does not resemble the predominant granular cytoplasmic tissue pattern (HPA tissue IHC: profile). In chromogenic IHC, inadequate blocking, excessive antibody concentration or detection background are general possibilities; compare a primary antibody omission control and optimize the workflow. |
| A known HPA positive cell population has little or no signal. | A negative slide alone does not establish absent CRY2. Confirm that the expected cells are present, then check antibody and detection performance with a positive control. HPA reports High staining in breast glandular cells and cerebellar granular layer cells, for example (HPA tissue IHC). No CRY2 specific fixation sensitivity or retrieval condition is supplied. |
💡Expected CRY2 appearanceCall a result convincing when identifiable HPA positive cells show High, granular cytoplasmic staining above background (HPA tissue IHC: positive cell levels and profile); diffuse staining or colour confined to unrelated structures warrants control review.
| Tissue and cell choice | Use an identifiable reported positive population to judge whether a slide worked: HPA lists High staining in adrenal glandular, bronchial respiratory epithelial and bone marrow hematopoietic cells (HPA tissue IHC). Its empty negative list does not identify a true negative tissue. UniProt describes expression across examined tissues, with highest levels in heart and skeletal muscle, but that statement is not an IHC intensity ranking (UniProt Q49AN0: tissue specificity). |
| Compartment and preparation | Tissue IHC is predominantly granular cytoplasmic (HPA tissue IHC: profile), while CRY2 can also occupy the nucleus (UniProt Q49AN0: subcellular location). Keep those observations distinct when reading paraffin sections: HPA's supported nuclear speckles and additional cytosol localization were observed by ICC-IF (HPA subcellular), so speckles are not a required tissue IHC feature. |
| IF/ICC Q&A: what pattern is reported? | HPA reports mainly nuclear speckles, with additional cytosol localization in ICC-IF (HPA subcellular: supported locations). Its images are listed for A-431, U-251MG and U2OS (HPA subcellular). That cell imaging result can inform interpretation, but it does not supply a paraffin IHC protocol or override the tissue IHC profile (HPA tissue IHC: granular cytoplasmic expression). |
| Isoforms and antibody coverage | UniProt lists 2 CRY2 isoforms (UniProt Q49AN0: isoforms). The payload gives no antibody epitope or isoform coverage, so do not assume that a negative result excludes both isoforms. It also gives no basis to predict an isoform specific compartment or cell pattern from this antibody. |
| Strength of validation | HPA marks the tissue IHC profile Supported, citing high consistency between antibody staining and RNA expression data (HPA tissue IHC: reliability). Antibody HPA037577 is listed as Supported for IHC and ICC (HPA antibodies). These labels support using the reported pattern as a reference; they do not make every unusual signal specific or establish a fixation effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported positive cells stain weakly or not at all. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Locate the expected cells and compare a concurrently processed positive control. Check the antibody, detection reagents and counterstain, then optimize retrieval and antibody concentration as general paraffin IHC steps; do not infer a CRY2 fixation effect from this failure. |
| Colour appears across the section, including spaces between cells. | Non-specific reagent deposition or insufficient suppression of endogenous detection activity is possible in chromogenic IHC; this appearance differs from the granular cytoplasmic HPA profile (HPA tissue IHC). | Inspect a primary antibody omission control, review blocking and washes, and adjust detection conditions. Score cell associated granules only after background is controlled; HPA supplies no target specific dilution for this section. |
| Only nuclei stain in a tissue section. | CRY2 can enter the nucleus (UniProt Q49AN0: subcellular location), but nuclear only tissue staining differs from HPA's predominant granular cytoplasmic IHC profile (HPA tissue IHC). | Verify nuclear boundaries with the counterstain and examine a reported positive population on the same run. Reassess specificity if cytoplasmic staining is absent; do not require the nuclear speckles observed in ICC-IF (HPA subcellular). |
| A structure or cell population outside HPA's examples stains strongly. | Cross-reactivity or endogenous chromogen activity may contribute, but HPA's positive list is selective and its negative list is empty (HPA tissue IHC); novelty alone cannot prove an artefact. | Check tissue morphology, compartment and the primary antibody omission control. Compare with a reported positive population and confirm unexpected staining independently before presenting it as CRY2 localization. |
| Signal varies among tissue samples or fields. | Cell composition can change what is visible; HPA assigns levels to specified cell populations, not uniformly to every cell in an organ (HPA tissue IHC: positive list). CRY2 participates in a circadian clock (UniProt Q49AN0: function), but the supplied IHC data do not quantify time dependent staining. | Score the same identifiable cell type and compartment across sections, record collection context if relevant, and compare slides processed together. Avoid attributing variation to circadian timing without matched sampling evidence. |
| ICC-IF shows speckles while the tissue IHC slide looks granular. | The patterns come from different preparations: ICC-IF mainly shows nuclear speckles with additional cytosol (HPA subcellular), whereas tissue IHC reports granular cytoplasm in a majority of tissues (HPA tissue IHC). | Interpret each preparation against its own HPA reference and controls. Use the ICC-IF result as localization context; judge the paraffin chromogenic section by the tissue IHC profile and its identifiable positive cells. |
Experiment evidence
IHC & IF images showing CRY2 staining
PB9576 · IHCIHC analysis of CRY2 using anti-CRY2 antibody (PB9576). CRY2 was detected in a paraffin-embedded section of rat liver tissue. Heat mediated
PB9576 · IHCIHC analysis of CRY2 using anti-CRY2 antibody (PB9576). CRY2 was detected in a paraffin-embedded section of mouse kidney tissue. Heat mediat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000121671-CRY2/tissue/cerebral+cortex#imid_8995610
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000121671-CRY2/tissue/colon#imid_8985024
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000121671-CRY2/tissue/liver#imid_8985009
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000121671-CRY2/tissue/kidney#imid_8985026
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037577; green = CRY2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037577; green = CRY2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRY2 IHC & IF
🧪Run bone marrow first and look for staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). HPA detects CRY2 in all 45 scored tissues, so no tissue is a validated negative; use no-primary and isotype controls, and treat any unstained cells on the positive slide as a background reference rather than proven CRY2-negative cells (HPA: no negative rows; detected in all 45 scored tissues).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CRY2 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 CRY2 in A-431, U-251MG, U2OS, with annotated localisation: Nuclear speckles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host-matched isotype control appropriate to the primary antibody’s clonality, and CRY2 knockout material or a validated peptide-block control where available (standard IHC practice). Block endogenous peroxidase in bone marrow before chromogenic detection, since hematopoietic cells can contribute peroxidase background (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the exact PB9576 paraffin-section caption does not state its fixative (PB9576 caption: fixative unreported). That caption used heat-mediated retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required (PB9576 caption: EDTA pH 8.0). The supplied evidence does not establish that frozen sections or IF are easier; IF/ICC images support nuclear-speckle and cytosolic localization, while endogenous peroxidase in bone marrow can complicate chromogenic IHC (HPA subcellular: nuclear speckles and cytosol; standard IHC practice).
HPA tissue IHC evidence for CRY2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High 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 | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CRY2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CRY2 IHC Tips
Use the PB9576 paraffin-section workflow as the starting point, then assess CRY2 staining by cell type, compartment and collection time (datasheet PB9576; HPA; UniProt Q49AN0).
Which retrieval conditions should I start with for CRY2 paraffin-section IHC?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9576). The reported workflow then used 10% goat serum, 1 μg/ml primary antibody overnight at 4°C, and chromogenic detection with DAB (datasheet PB9576). If staining is weak, compare heating durations on matched sections before trying another retrieval buffer as a fallback (standard IHC practice). Keep antibody concentration, detection and development conditions constant while comparing retrieval, so a change in signal has a clearer cause (standard IHC practice). Examine both cytoplasmic and nuclear staining during optimisation because CRY2 occupies both compartments (UniProt Q49AN0).
How should I troubleshoot fixation-related loss of CRY2 staining?
Target-specific CRY2 sensitivity to fixation is unknown because the PB9576 paraffin-section caption does not state a fixative (datasheet PB9576). Record the fixative, fixation duration and tissue processing history for each specimen before comparing stain intensity (standard IHC practice). Compare sections with documented, similar processing using the same EDTA retrieval at pH 8.0 and 1 μg/ml primary antibody (datasheet PB9576; standard IHC practice). If a processing group stains weakly, include a consistently stained reference section in the same run to check assay performance (standard IHC practice). Do not assign the difference to CRY2-specific fixation sensitivity without a controlled comparison (standard IHC practice).
Should CRY2 staining be nuclear or cytoplasmic in tissue sections?
Assess both compartments: CRY2 is annotated in the cytoplasm and nucleus, with nuclear translocation through interactions with clock proteins including PER2 or BMAL1 (UniProt Q49AN0). Tissue IHC shows granular cytoplasmic expression in a majority of tissues, while subcellular imaging supports nuclear speckles and additional cytosolic localisation (HPA tissue IHC; HPA subcellular). Score nuclear and cytoplasmic staining separately rather than requiring one pattern in every cell (standard IHC practice; UniProt Q49AN0). Check that apparent nuclear signal remains within counterstained nuclei and that cytoplasmic granules follow cell boundaries (standard IHC practice). Compare matched collection times because CRY2 participates in the circadian oscillator (UniProt Q49AN0).
Can this stain distinguish CRY2 isoforms or phosphorylation states?
CRY2 has 2 annotated isoforms, but the supplied PB9576 tissue-IHC caption does not identify the antibody epitope (UniProt Q49AN0; datasheet PB9576). Therefore, treat chromogenic signal as CRY2 staining without assigning it to an individual isoform (standard IHC interpretation). The protein includes a photolyase/cryptochrome domain at residues 22–151 and annotated phosphoserines at positions including 90, 266, 299, 554 and 558 (UniProt Q49AN0). Those annotations do not establish whether processing or phosphorylation changes antibody binding when its epitope is unknown (standard IHC interpretation). Use independently characterised isoform-specific or modification-specific reagents before making either claim (standard IHC practice).
How can I investigate CRY2 localisation by multiplex immunofluorescence?
Treat IF/ICC as a separate assay and establish its staining conditions independently of the PB9576 paraffin-section IHC workflow (standard IF practice; datasheet PB9576). Pair CRY2 with a validated marker for the expected cell type, such as the glandular-cell population reported in adrenal gland IHC, and include a nuclear counterstain (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore when tissue autofluorescence interferes with shorter-wavelength channels, and inspect unstained controls in each channel (standard IF practice). CRY2 has no transmembrane segment and occupies nuclear and cytosolic compartments, so optimise permeabilisation for access to both; the antibody epitope is unspecified (UniProt Q49AN0; datasheet PB9576).
What should I check when CRY2 DAB staining appears diffuse or widespread?
The PB9576 example used 10% goat serum blocking, a biotinylated secondary antibody, a streptavidin-biotin detection complex and DAB (datasheet PB9576). Include a no-primary control and check endogenous peroxidase blocking before attributing diffuse DAB colour to CRY2 (standard IHC practice). With biotin-based detection, assess endogenous biotin as another possible background source using an appropriate control (datasheet PB9576; standard IHC practice). Compare primary-antibody dilution, wash stringency and DAB development across matched sections rather than changing them simultaneously (standard IHC practice). Judge residual signal against cell boundaries and the reported granular cytoplasmic tissue pattern (HPA tissue IHC; standard IHC practice).
How should I quantify CRY2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring because tissue IHC reports predominantly granular cytoplasmic staining, while CRY2 can also localise to nuclei (HPA tissue IHC; UniProt Q49AN0). For an intensity measure, record the percentage of cells at each intensity and calculate an H-score from 0–300; report nuclear and cytoplasmic scores separately (standard IHC practice). Alternatively, report percentage-positive cells or positive-cell density per mm² within a predefined tissue area (standard IHC practice). Normalise cell counts to the number of eligible cells or measured area and keep threshold, illumination and DAB development consistent (standard IHC practice). Record collection time because CRY2 is a circadian-clock component (UniProt Q49AN0).
How can I distinguish credible CRY2 staining from artefact?
Look for cell-associated signal with assessable cytoplasmic or nuclear localisation; CRY2 occupies both compartments, and tissue IHC commonly shows granular cytoplasmic staining (UniProt Q49AN0; HPA tissue IHC). Compare stained cells with the expected cell population, such as glandular cells in adrenal gland, rather than interpreting colour without tissue context (HPA tissue IHC; standard IHC practice). Discount staining confined to section edges, damaged or necrotic regions, or the no-primary control (standard IHC practice). Check endogenous peroxidase when DAB persists without primary antibody, and consider endogenous biotin with the reported biotin-based detection method (datasheet PB9576; standard IHC practice). Interpret differences between specimens alongside collection time and processing records (UniProt Q49AN0; standard IHC practice).
Boster reagents
Best CRY2 / Cryptochrome-2 IHC Antibodies
PB9576 has real CRY2 IHC images from paraffin sections of rat liver, mouse kidney and human heart (catalog IHC image captions).
Real IHC data
Anti-CRY2 Antibody ®
Cat # PB9576
PB9576 is listed for IHC in human, mouse and rat samples (catalog: applications and reactivity). Its IHC captions show staining in rat liver, mouse kidney and human heart paraffin sections (PB9576 IHC image captions).
Which to pick: For tissue IHC, choose PB9576 for paraffin sections; its rat liver and mouse kidney captions use 1 μg/ml, while its human heart caption uses 2 μg/ml (PB9576 IHC image captions). PB9576 is also the cross-species choice for human, mouse and rat IHC (catalog: applications and reactivity). No IF/ICC option is supported by this payload (catalog: applications and IF images); the IHC captions do not report the fixative (PB9576 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q49AN0 (CRY2_HUMAN, Cryptochrome-2).
- Human Protein Atlas. CRY2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CRY2 subcellular location (ICC-IF): Mainly localized to the nuclear speckles. In addition localized to the cytosol..
- Human Protein Atlas. CRY2 antibody validation summary (1 antibodies).
- Aberrant rhythmic expression of cryptochrome2 regulates the radiosensitivity of rat gliomas. Oncotarget 2017 — PMC5652816.
- Circadian Clock Gene CRY2 Degradation Is Involved in Chemoresistance of Colorectal Cancer. Molecular cancer therapeutics 2015 — PMC4458447.
- Uncovering the Penile Clock: Expression of Molecular Clock Proteins in Human Penile Cavernous Tissue. The world journal of men's health 2024 — PMC11439802.
- Suppression of circadian clock protein cryptochrome 2 promotes osteoarthritis. Osteoarthritis and cartilage 2020 — PMC7476803.
- PubMed PMID:8909283 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRY2 (Cryptochrome-2). Expected localisation Granular cytoplasm (HPA tissue IHC); nucleus possible (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9576). Validated IHC antibody: PB9576 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (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 rat liver tissue; fixative not specified (datasheet PB9576); 2. Fixation: Image fixative and duration unreported (datasheet PB9576); 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 PB9576); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9576); 8. Primary antibody: Rabbit anti-CRY2, 0.5-1μg/ml (datasheet PB9576); 9. Primary incubation: Overnight at 4 °C (datasheet PB9576). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9576); Caveat: Sampling time may affect intensity comparisons (UniProt); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Circadian feedback regulates CRY2 (UniProt). Evidence: UniProt Q49AN0, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nuclear speckles. In addition localized to…, Datasheet PB9576, PMC5652816, PMC4458447. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRY2/immunohistochemistry-ihc-guide.