CRY1 / Cryptochrome-1 · IHC design guide

Design Immunohistochemistry for CRY1

Plan chromogenic CRY1 IHC in paraffin sections with the catalog antibody at 0.5–1 μg/mL (datasheet: PB9540). Assess cytoplasmic and nuclear staining against the tissue profile and record sampling time for this circadian regulator (HPA tissue IHC; UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for CRY1 (IHC for CRY1): expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC), antibody PB9540, validated IHC image, and IHC protocol steps — Printable **CRY1** IHC protocol sheet — expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC), antibody PB9540, controls and protocol steps. Open the full CRY1 IHC guide →

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CRY1 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	Cytoplasmic and nuclear tissue staining (HPA tissue IHC)
	Staining pattern	Widespread cytoplasmic and nuclear cell staining (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet PB9540)
	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	Keep fixation consistent across samples (standard IHC practice; not target-specific)
	Caveat	Staining and RNA levels show low consistency (HPA tissue IHC)
	Regulation	Circadian cycling (UniProt)
	Isoform / epitope	0 annotated isoforms; one full-length chain (UniProt)

Section 1

Recommended CRY1 IHC & IF Protocols

The catalog antibody protocol is accompanied by published CRY1 IHC methods for cartilage and colorectal tissue (PMC7476803; PMC3634012).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat intestine tissues; fixative not specified (datasheet PB9540)
Fixation	Image fixative and duration unreported (datasheet PB9540); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet PB9540)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9540)
Primary antibody	Rabbit anti-CRY1, 0.5-1μg/ml (datasheet PB9540)
Primary incubation	Overnight at 4 °C (datasheet PB9540)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9540)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CRY1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic and nuclear expression. No signal in the no-primary control.

💡Decision noteStart with heat retrieval in citrate at pH 6, as specified for the catalog antibody and the colorectal tissue protocol (datasheet PB9540; PMC3634012).

Section 2

What Is the Expected CRY1 Staining Pattern?

CRY1 is found in the cytoplasm and nucleus and can move into the nucleus through interactions with clock proteins (UniProt Q16526). In paraffin-section IHC, expect staining across many tissues, including strong staining in the cell populations listed by HPA (HPA: ubiquitous cytoplasmic and nuclear expression; selected cells High). Interpret intensity cautiously: HPA rates the antibody Approved but reports low consistency between staining and RNA expression (HPA: reliability description). CRY1 has no transmembrane segment (UniProt Q16526 topology).

What am I looking at on my slide?
Cytoplasmic and nuclear staining in adrenal glandular cells, for example.	This fits CRY1 localisation and a listed high-staining population (UniProt Q16526 subcellular location; HPA: High in adrenal glandular cells). Nuclear-to-cytoplasmic balance may differ between cells because CRY1 can translocate with clock proteins (UniProt Q16526).
Staining restricted to an unexpected compartment, with no convincing cytoplasmic or nuclear signal.	Treat the pattern as suspect and check morphology and controls: the established IHC-compatible locations are cytoplasm and nucleus (UniProt Q16526; HPA: ubiquitous cytoplasmic and nuclear expression). An unusual pattern alone cannot establish a new CRY1 location.
Strong staining in a cell population outside the listed HPA high-staining populations.	Check cell identity and the no-primary control for cross-reactivity or endogenous chromogen activity. HPA describes broad expression and lists selected high-staining cells, so an unlisted population is not automatically CRY1-negative (HPA: tissue IHC profile and positive list).
Diffuse colour over tissue, stroma, and empty areas rather than defined cellular staining.	Background obscures compartment assignment. Review blocking, washes, antibody concentration, and detection controls as general IHC checks; HPA reports a cellular cytoplasmic and nuclear pattern, not diffuse field-wide colour (HPA: tissue IHC profile).
No cellular signal in a section of a listed high-staining tissue.	First check that the relevant cells are present and preserved, then review retrieval, antibody and detection steps using run controls. HPA lists High staining in several populations, but its Approved rating has low staining–RNA consistency (HPA: positive list; reliability description).

💡Expected CRY1 appearanceCall positive when defined cytoplasmic and/or nuclear colour is visible in identifiable cells, with strong signal plausible in HPA-listed High populations; diffuse field-wide colour or staining without cellular structure is suspect (UniProt Q16526 subcellular location; HPA: tissue IHC profile and positive list).

How each factor affects the staining
Cell population and tissue context (HPA: tissue IHC positive list).	HPA reports High staining in adrenal, appendix, and cervical glandular cells; marrow hematopoietic cells; bronchial respiratory epithelial cells; caudate and cortical glia; and cerebellar granular-layer cells. Confirm the relevant population on the section before judging a weak field (HPA: High in listed cells).
Compartment and clock-protein interactions (UniProt Q16526).	CRY1 occupies cytoplasm and nucleus and moves into the nucleus through interactions including PER2 or BMAL1 (UniProt Q16526 subcellular location). Interpret a change in their relative staining cautiously; the supplied sources give no sampling-time threshold for IHC scoring.
Strength of reference evidence (HPA: Approved; low staining–RNA consistency).	The listed antibody, CAB018762, is Approved for IHC; the supplied record does not rate it Enhanced (HPA: antibody validation). HPA also reports low consistency between staining and RNA expression, so a matching pattern supports interpretation but does not establish antibody specificity by itself (HPA: reliability description).
Intracellular topology (UniProt Q16526 topology and processing).	CRY1 has no transmembrane segment, signal peptide, or propeptide; its annotated chain spans residues 1–586 (UniProt Q16526). Judge staining as cellular rather than membrane-only. These annotations do not establish an antigen-retrieval setting or a fixation effect.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known high-staining cells appear blank (HPA: High in listed cells).	The expected cells may be absent from the section, or the IHC run may have failed; the image alone cannot distinguish these causes.	Verify cell identity and section integrity, then check a positive run control, retrieval, primary incubation, and chromogenic detection. Record the HPA reliability caveat when interpreting the result (HPA: Approved; low staining–RNA consistency).
Colour appears without primary antibody.	Endogenous detection activity or nonspecific chromogen deposition is possible (general IHC practice).	Compare the no-primary control and apply the detection system's appropriate endogenous-activity block; reassess only cellular signal above that control (general IHC practice).
Most of the field has diffuse colour.	Nonspecific binding, inadequate washing, or excessive antibody or detection signal may obscure the expected cellular pattern (general IHC practice; HPA: cytoplasmic and nuclear profile).	Review blocking, washes, antibody titration, and detection exposure against control sections; score only interpretable cells (general IHC practice).
Signal seems confined to cell borders.	A border-only pattern conflicts with the reported cytoplasmic and nuclear distribution and CRY1's lack of a transmembrane segment (HPA: tissue IHC profile; UniProt Q16526 topology).	Check morphology, focus, and control staining before assigning a compartment; confirm any cellular signal extends into cytoplasm or nucleus (HPA: tissue IHC profile).
Nuclear intensity varies among otherwise comparable cells.	CRY1 can translocate to the nucleus through clock-protein interactions; the supplied sources do not establish a phase-specific IHC intensity cutoff (UniProt Q16526).	Score nuclear and cytoplasmic compartments separately and keep sampling conditions consistent when comparing sections (general IHC practice).
IF/ICC Q&A: Should the IHC pattern match fluorescent cell images exactly?	IF/ICC has a separate evidence context: HPA reports nucleoplasm as supported, and nuclear membrane and microtubules as approved ICC-IF locations (HPA: subcellular summary).	Use those locations as context, then assess paraffin IHC against its own cellular pattern and controls; this IHC guide supplies no IF/ICC protocol (HPA: tissue IHC profile; subcellular summary).

Source: UniProt Q16526 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CRY1 staining

IHC analysis of CRY1 using anti-CRY1 antibody (PB9540). CRY1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CRY1 Antibody (PB9540) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9540 · IHCIHC analysis of CRY1 using anti-CRY1 antibody (PB9540). CRY1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediat

IHC analysis of CRY1 using anti-CRY1 antibody (PB9540). CRY1 was detected in paraffin-embedded section of mouse intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CRY1 Antibody (PB9540) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9540 · IHCIHC analysis of CRY1 using anti-CRY1 antibody (PB9540). CRY1 was detected in paraffin-embedded section of mouse intestine tissues. Heat medi

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000008405-CRY1/tissue/cerebral+cortex#imid_4719556

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000008405-CRY1/tissue/colon#imid_4719466

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000008405-CRY1/tissue/liver#imid_4719467

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000008405-CRY1/tissue/kidney#imid_4719468

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB018762; green = CRY1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for CRY1 IHC & IF

🧪Run adrenal gland first; its glandular cells should stain for CRY1 (HPA: High in adrenal gland glandular cells). HPA detects CRY1 in all 45 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and expect cells without specific staining on the positive slide to show counterstain without chromogen above background (HPA: no negative rows; standard IHC practice).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: CRY1 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 CRY1 in A-431, HEK293, U2OS, with annotated localisation: Nucleoplasm (supported), Nuclear membrane (approved), Microtubules (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide; a concentration-matched rabbit control IgG matched to the primary’s isotype and clonality where known; and CRY1 knockout tissue or a peptide-blocked primary when the immunizing peptide is available (PB9540 caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check for endogenous biotin before using the caption’s biotin-based DAB detection on adrenal tissue (PB9540 caption: biotinylated secondary, streptavidin-biotin complex and DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected PB9540 paraffin-section caption does not state the fixative (PB9540 caption). That caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required (PB9540 caption). The evidence does not establish whether frozen sections or IF are easier; HPA provides ICC-IF images in A-431, HEK293 and U2OS, while biotin-based chromogenic detection on adrenal sections warrants an endogenous-biotin check (HPA: subcellular images; PB9540 caption; standard IHC practice).

HPA tissue IHC evidence for CRY1

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	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Caudate	Glial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: CRY1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced CRY1 IHC Tips

Troubleshoot CRY1 staining in paraffin sections by checking retrieval, compartment-specific signal, detection controls, and the timing of tissue collection.

What retrieval should I start with for weak CRY1 staining?

Use heat-mediated citrate buffer at pH 6 for 20 minutes as the starting antigen retrieval condition (datasheet PB9540; PB9540 tissue-IHC caption). The documented paraffin-section workflow detected CRY1 in rat intestine after that treatment, followed by overnight incubation at 4°C with 1 μg/mL primary antibody (PB9540 tissue-IHC caption). If staining remains weak, check section adherence, heating consistency, and primary-antibody coverage before changing retrieval conditions (standard IHC practice). Compare any adjusted heating time with the documented condition on matched sections, because stronger retrieval can also damage morphology or raise background (standard IHC practice).

Could fixation explain weak or uneven CRY1 staining?

The PB9540 paraffin-section caption does not state a fixative, so CRY1-specific sensitivity to fixation is unknown (PB9540 tissue-IHC caption). Record the fixative, fixation duration, section thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). On matched sections, compare the documented pH 6 citrate retrieval for 20 minutes with a carefully controlled adjustment if morphology and signal warrant it (PB9540 tissue-IHC caption; standard IHC practice). Examine whether weak staining tracks processing batches or tissue edges, and use a consistently processed reference section to distinguish handling variation from a possible biological difference (standard IHC practice).

Should CRY1 stain nuclei, cytoplasm, or both?

Score nuclear and cytoplasmic staining separately: CRY1 is reported in both compartments and can enter the nucleus through interactions with clock proteins such as PER2 or BMAL1 (UniProt Q16526 subcellular location). HPA describes ubiquitous cytoplasmic and nuclear tissue staining, although its tissue-IHC reliability is marked Approved with low consistency against RNA expression (HPA tissue IHC). Check the counterstain and cell boundaries before assigning a faint signal to either compartment (standard IHC practice). If one compartment predominates, record collection time and processing batch before treating the pattern as altered CRY1 trafficking, because CRY1 participates in the circadian oscillator (UniProt Q16526 function; standard IHC practice).

How can I assess an unexpected CRY1 staining pattern without epitope information?

The supplied record lists 0 CRY1 isoforms and a 586-residue chain, so an isoform explanation is unsupported here (UniProt Q16526 processing and isoforms). CRY1 has no transmembrane segment, while reported phosphoserines include residues 71, 247, 280, and 568 (UniProt Q16526 topology and modified residues). Request the catalog antibody’s immunogen or mapped epitope before proposing that a modification, retrieval condition, or protein interaction masks its binding site (standard IHC practice). Compare serial sections using a documented positive reference and primary-omission control; do not assign an unexpected compartment to a hypothetical epitope effect without independent validation (standard IHC practice).

How should I adapt CRY1 localisation checks to multiplex IF?

Treat IF as a separate validation workflow: the documented PB9540 tissue example uses chromogenic detection in a paraffin section (PB9540 tissue-IHC caption). Multiplex CRY1 with a validated marker for the cell population being examined, and include a nuclear counterstain to distinguish nuclear from cytoplasmic signal (standard IF practice; UniProt Q16526 subcellular location). Choose fluorophores after inspecting unstained tissue for autofluorescence, favouring channels with clearer signal separation and appropriate single-stain controls (standard IF practice). Because CRY1 has no transmembrane segment and occupies intracellular compartments, establish permeabilisation for cytoplasmic or nuclear access after checking the antibody’s epitope information; HPA reports nucleoplasmic, nuclear-membrane, and microtubule localisation in its subcellular dataset (UniProt Q16526 topology; HPA subcellular; standard IF practice).

How do I separate CRY1 staining from chromogenic background?

Start with a primary-omission section and inspect endogenous peroxidase activity, nonspecific secondary binding, and precipitated chromogen (standard IHC practice). The documented PB9540 workflow used 10% goat serum blocking, a biotinylated secondary antibody, a streptavidin–biotin complex, and DAB development (PB9540 tissue-IHC caption). If diffuse staining persists, compare a controlled primary-antibody dilution series around the documented 1 μg/mL condition and check whether background follows the detection reagents (PB9540 tissue-IHC caption; standard IHC practice). Assess tissue edges, folds, and damaged areas separately from intact cells, while retaining nuclear and cytoplasmic CRY1 as plausible compartments (standard IHC practice; UniProt Q16526 subcellular location).

What should I measure when comparing CRY1 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, then report nuclear and cytoplasmic CRY1 separately because both locations are documented (UniProt Q16526 subcellular location; standard IHC practice). Use percentage of positive cells or a compartment-specific H-score with fixed intensity thresholds, and report the number of evaluable cells or sampled area (standard IHC practice). Normalise each result to the relevant cell count or tissue area, such as positive cells per mm², and exclude folds, necrosis, and section edges using preset rules (standard IHC practice). Keep staining batches and collection times comparable, since CRY1 is a circadian-clock component and apparent intensity differences need temporal context (UniProt Q16526 function; standard IHC practice).

When is an unusual CRY1 pattern likely to be artefactual?

A credible CRY1 result should resolve to intact cells with interpretable nuclear or cytoplasmic staining, consistent with its reported locations (UniProt Q16526 subcellular location; standard IHC practice). Staining confined to section edges, folds, necrotic material, or a primary-omission control favours processing or detection artefact (standard IHC practice). Verify the apparent cell identity on the counterstained section; HPA reports high staining in several cell populations but also notes low consistency between antibody staining and RNA expression (HPA tissue IHC). Check endogenous peroxidase and biotin-related background when interpreting the documented biotin-based DAB workflow, and compare matched collection times before attributing a compartment shift to circadian biology (PB9540 tissue-IHC caption; UniProt Q16526 function; standard IHC practice).

Boster reagents

Best CRY1 / Cryptochrome-1 IHC Antibodies

PB9540 is an IHC-validated anti-CRY1 antibody with paraffin-section images from rat and mouse intestine (PB9540 IHC captions). The catalog lists human, mouse and rat reactivity (PB9540 catalog).

Real IHC data

Anti-Cryptochrome I/CRY1 Antibody ®

Cat # PB9540

View product →

PB9540 will render with its IHC figure from paraffin-embedded rat intestine (PB9540 figure caption). Additional PB9540 IHC images document mouse intestine, and the catalog lists human, mouse and rat reactivity (PB9540 IHC captions; PB9540 catalog).

Which to pick: For tissue IHC, choose PB9540: its paraffin-section captions describe citrate retrieval at pH 6 and staining at 1 μg/ml (PB9540 IHC captions). For cross-species work, PB9540 lists human, mouse and rat reactivity, while the supplied IHC images show rat and mouse tissue (PB9540 catalog; PB9540 IHC captions). No IF/ICC application or dilution is supplied, so this payload supports no IF/ICC recommendation; clonality and tissue fixative are unreported (PB9540 catalog; PB9540 IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q16526 (CRY1_HUMAN, Cryptochrome-1).
Human Protein Atlas. CRY1 tissue IHC expression (reliability: Approved).
Human Protein Atlas. CRY1 subcellular location (ICC-IF): Localized to the nucleoplasm, nuclear membrane and microtubules..
Human Protein Atlas. CRY1 antibody validation summary (1 antibodies).
Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals. Scientific reports 2016 — PMC4761878.
Full-Length Cryptochrome 1 in the Outer Segments of the Retinal Blue Cone Photoreceptors in Humans and Great Apes Suggests a Role Beyond Transcriptional Repression. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2025 — PMC12023722.
Suppression of circadian clock protein cryptochrome 2 promotes osteoarthritis. Osteoarthritis and cartilage 2020 — PMC7476803.
Cryptochrome 1 overexpression correlates with tumor progression and poor prognosis in patients with colorectal cancer. PloS one 2013 — PMC3634012.
PubMed PMID:8909283 — UniProt-cited evidence.
PubMed PMID:8921389 — UniProt-cited evidence.
PubMed PMID:8600518 — UniProt-cited evidence.