CRY1 / Cryptochrome-1 · IHC design guide

Design Immunohistochemistry for CRY1

Plan chromogenic CRY1 IHC on paraffin sections using the IHC-validated antibody (datasheet PB9540). Score cytoplasmic and nuclear staining against the reported tissue pattern (HPA tissue IHC), accounting for circadian expression (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 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 conditions consistent across sections (standard IHC practice; not target-specific)
	Caveat	Staining has low consistency with RNA expression (HPA tissue IHC)
	Regulation	Expression follows ~24 h rhythms (UniProt)
	Isoform / epitope	No annotated isoforms or cleavage; one 1–586 chain (UniProt)

Section 1

Recommended CRY1 IHC & IF Protocols

The catalog antibody uses citrate pH 6 retrieval (datasheet PB9540). Published IHC protocols add cartilage and colorectal tissue examples (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 citrate pH 6 heat retrieval (datasheet PB9540); the colorectal protocol specifies 95°C for 25 minutes (PMC3634012).

Section 2

What Is the Expected CRY1 Staining Pattern?

CRY1 staining may be cytoplasmic and nuclear (UniProt Q16526; HPA tissue IHC). In paraffin sections, HPA reports high staining in adrenal glandular cells, bronchial respiratory epithelial cells, bone marrow hematopoietic cells and cerebral cortical glial cells (HPA tissue IHC). Its tissue profile is described as ubiquitous, but the IHC assessment is Approved with low consistency between staining and RNA expression (HPA tissue IHC). CRY1 has no transmembrane segment (UniProt Q16526 topology).

What am I looking at on my slide?
Cytoplasmic and nuclear chromogen appears in the expected cells of an HPA high-staining tissue (HPA tissue IHC).	This fits the reported tissue pattern (HPA tissue IHC). Compare nuclei with the counterstain and assess signal within identifiable cells (standard IHC practice). Neither compartment must dominate every cell: CRY1 can move into the nucleus through interactions with clock proteins (UniProt Q16526).
Signal outlines extracellular spaces or appears only at the apical surface, with little intracellular staining.	Treat this as a localization mismatch requiring investigation: CRY1 is reported in cytoplasm and nucleus and has no transmembrane segment (UniProt Q16526). Check tissue morphology, staining controls and detection reagents before assigning such deposits to CRY1 (standard IHC practice).
Strong signal appears in cells outside the anticipated population while the expected population is weak.	Cross-reactivity or endogenous detection activity is possible (standard IHC practice). Cell identity alone cannot establish a false positive because HPA describes CRY1 tissue expression as ubiquitous; compare the compartments and a negative reagent control (HPA tissue IHC; standard IHC practice).
Chromogen spreads across stroma, lumina or most cells without clear intracellular boundaries.	Diffuse background obscures whether cytoplasmic or nuclear CRY1 is present (UniProt Q16526; standard IHC practice). Examine a control lacking primary antibody; if the same deposit persists, investigate detection chemistry and washing before interpreting staining intensity (standard IHC practice).
No convincing intracellular signal appears in bronchial respiratory epithelium or adrenal glandular cells.	Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.

💡Expected CRY1 appearanceCall positive staining when identifiable HPA high-staining cells show clear cytoplasmic and/or nuclear chromogen above local background; diffuse extracellular deposits or signal confined to an unsupported compartment should prompt control checks (HPA tissue IHC; UniProt Q16526; standard IHC practice).

How each factor affects the staining
Compartment and clock context (UniProt Q16526)	CRY1 is reported in cytoplasm and nucleus, and interaction with PER2 or BMAL1 can promote nuclear translocation (UniProt Q16526). A different nuclear-to-cytoplasmic balance can therefore be biologically plausible; this record does not specify an expected staining ratio for a given collection time (UniProt Q16526).
Choice of tissue and cell population (HPA tissue IHC)	HPA reports high staining in adrenal glandular cells, bone marrow hematopoietic cells, bronchial respiratory epithelium and glial cells in caudate and cerebral cortex (HPA tissue IHC). Its supplied list contains no negative or low-staining tissue, so it does not support naming a CRY1-negative tissue control (HPA tissue IHC).
Confidence of the tissue pattern (HPA tissue IHC)	The tissue IHC assessment is Approved, while HPA notes low consistency between antibody staining and RNA expression data (HPA tissue IHC). Use the reported pattern as a reference for interpretation, and resolve unexpected staining with controls rather than treating the map as definitive for every cell (HPA tissue IHC; standard IHC practice).
Antibody validation scope (HPA antibodies)	CAB018762 is a rabbit polyclonal antibody listed as Approved for IHC and ICC (HPA antibodies). The supplied antibody record does not label its IHC validation Enhanced; an Approved result alone should not be described as independently reproduced by multiple antibodies (HPA antibodies).
Intracellular target and epitope information (UniProt Q16526)	CRY1 has no signal peptide, propeptide, transmembrane segment or annotated isoforms in the supplied record (UniProt Q16526). Its antibody epitope is unspecified here, so these features cannot establish a CRY1-specific retrieval setting or predict how fixation will affect staining (UniProt Q16526; HPA antibodies).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
An HPA high-staining population is blank in an otherwise readable section (HPA tissue IHC).	Possible assay failure; the supplied sources do not establish a CRY1-specific fixation effect (standard IHC practice; HPA tissue IHC).	Confirm that a positive control stains, then review antibody application, retrieval and detection against the validated IHC procedure (standard IHC practice).
Stain is widespread but cell boundaries and nuclei cannot be assessed.	Background from the staining workflow may mask intracellular signal (standard IHC practice).	Compare a control lacking primary antibody, check washing and blocking, and score only distinguishable cellular staining above background (standard IHC practice).
Brown deposit persists in the control without primary antibody.	Endogenous enzyme activity or detection-reagent background is possible with chromogenic detection (standard IHC practice).	Check the detection system and its appropriate endogenous-activity block; repeat the control before assigning the deposit to CRY1 (standard IHC practice).
Only surface or extracellular deposits remain after background is reduced.	That distribution conflicts with reported cytoplasmic and nuclear CRY1 localization (UniProt Q16526; HPA tissue IHC).	Recheck morphology and controls, then compare intracellular staining in an HPA high-staining cell population (standard IHC practice; HPA tissue IHC).
An unexpected cell population stains strongly.	Cross-reactivity is possible, but CRY1 is described as ubiquitously expressed, so cell identity alone is inconclusive (standard IHC practice; HPA tissue IHC).	Confirm the cell type and compartment, compare the control without primary antibody, and seek independent validation if the claim depends on that population (standard IHC practice).
Q: How should an IF/ICC image be interpreted alongside this IHC result?	HPA reports nucleoplasm as supported, with nuclear membrane and microtubules approved in ICC-IF; these are modality-specific observations (HPA subcellular).	A: Compare nuclear and cytoplasmic localization cautiously across modalities. Use this page's paraffin-section chromogenic guidance for IHC; consult the separate IF/ICC guide for its workflow (UniProt Q16526; HPA subcellular).

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 bone marrow first and assess CRY1 staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). HPA detects CRY1 in all 45 scored tissues, so there is no documented negative tissue; use no-primary and isotype controls, and treat cells without visible signal on the positive slide as internal background references rather than proven CRY1-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: 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 and a rabbit IgG isotype control matched to the primary antibody’s clonality where known, plus CRY1 knockout material or a validated peptide-block control (PB9540 caption: rabbit anti-CRY1 antibody; standard IHC controls). Block endogenous peroxidase and check for endogenous biotin in bone marrow because the caption uses biotinylated secondary antibody, SABC and DAB; hematopoietic cells can contain endogenous peroxidase (PB9540 caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9540 paraffin-section caption does not state the fixative (PB9540 caption: fixative unreported). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, which provides a starting condition but does not establish that retrieval is required (PB9540 caption: citrate retrieval); endogenous peroxidase in bone marrow may complicate DAB interpretation (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier for CRY1 (HPA: ICC-IF images in A-431, HEK293 and U2OS; PB9540 caption: paraffin-section IHC).

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, controls and scoring before interpreting differences between samples.

Which retrieval conditions should I try first for weak CRY1 staining?

Start with heat-mediated citrate buffer at pH 6 for 20 minutes on paraffin sections (datasheet PB9540). The PB9540 rat intestine image used these conditions, followed by 1 μg/mL primary antibody overnight at 4 °C (datasheet PB9540). If staining is weak, compare a modestly longer or shorter heating time on matched sections while keeping detection conditions fixed (standard IHC practice). Include an adjacent section processed without primary antibody to reveal retrieval-associated background (standard IHC practice). Record tissue preservation alongside signal, since excessive heating can impair morphology and make compartment scoring unreliable (standard IHC practice).

Could fixation explain weak or patchy CRY1 staining?

CRY1-specific fixation sensitivity is unknown: the PB9540 paraffin-section caption does not report a fixative (datasheet PB9540). Record the fixative, fixation duration, processing history and section age for each specimen before comparing staining intensity (standard IHC practice). When possible, stain similarly processed sections together using citrate retrieval at pH 6 for 20 minutes as the documented starting condition (datasheet PB9540). Compare morphology and the distribution of staining across each section; uneven preservation can complicate interpretation (standard IHC practice). Do not assign a weak result to a CRY1-specific fixation effect without a controlled fixation comparison using the same antibody and tissue (standard IHC practice).

Should CRY1 stain nuclei, cytoplasm or both in IHC?

Score nuclear and cytoplasmic staining separately because CRY1 is reported in both compartments (UniProt Q16526 subcellular location). Its movement into the nucleus involves interactions with clock proteins including PER2 or BMAL1, so a fixed section may capture differing distributions (UniProt Q16526 subcellular location). HPA describes ubiquitous cytoplasmic and nuclear tissue staining, while its cell imaging places CRY1 in the nucleoplasm, nuclear membrane and microtubules (HPA tissue IHC; HPA subcellular). Use the counterstain and preserved cell boundaries to assign compartments consistently across matched sections (standard IHC practice). Treat isolated extracellular deposits as suspect and check the no-primary control before calling them CRY1 (standard IHC practice).

How should epitope uncertainty affect CRY1 IHC troubleshooting?

The supplied record lists 0 isoforms, but gives no antibody epitope, so isoform-selective staining cannot be inferred for PB9540 (UniProt Q16526 isoforms; datasheet PB9540). CRY1 has no transmembrane segment or annotated glycosylation sites, while reported phosphorylation includes residues 71, 247, 280 and 568 (UniProt Q16526 topology, glycosylation and modified residues). Those annotations do not establish whether any modification masks this antibody's epitope in a section (standard IHC interpretation). Keep retrieval and detection matched when comparing specimens, and document any change in nuclear versus cytoplasmic signal (standard IHC practice). If specificity remains uncertain, use an independently validated CRY1 antibody or a validated loss-of-target control where available (standard IHC practice).

How can IF help assess the CRY1 IHC localisation pattern?

Use IF as a separate localisation check, since CRY1 can appear in both nucleus and cytoplasm (UniProt Q16526 subcellular location). Multiplex a CRY1 channel with a validated marker for the cell population being examined, and include a nuclear counterstain to assign signals to individual cells (standard IF practice). Select fluorophores after checking unstained tissue for autofluorescence; a far-red channel may improve separation if shorter wavelengths show strong background (standard IF practice). CRY1 lacks a transmembrane segment, so permeabilisation should permit antibody access to intracellular epitopes while preserving compartment boundaries (UniProt Q16526 topology; standard IF practice). Optimise IF fixation and permeabilisation directly for that assay rather than assuming the paraffin IHC image establishes those conditions (datasheet PB9540; standard IF practice).

What should I check when CRY1 DAB staining is diffuse?

First compare the stained section with a no-primary control and inspect whether color follows cells or also covers edges and damaged areas (standard IHC practice). The PB9540 example used 10% goat serum, biotinylated secondary antibody, a streptavidin-biotin complex and DAB detection (datasheet PB9540). Check secondary-only background, endogenous biotin where relevant, and peroxidase blocking as routine controls for this detection chemistry (standard IHC practice). Titrate primary antibody around the documented 1 μg/mL condition if diffuse staining obscures nuclei and cytoplasm (datasheet PB9540; standard IHC practice). Keep development time and counterstaining consistent across comparison slides so background differences are easier to recognise (standard IHC practice).

How should I quantify CRY1 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because CRY1 is reported in both nucleus and cytoplasm (UniProt Q16526 subcellular location). For each compartment, report the percentage of positive cells and an intensity-weighted H-score from 0–300, or count positive cells per mm² when cell density is the main outcome (standard IHC practice). Normalise counts to the annotated tissue area and compare like cell populations, section regions and staining batches (standard IHC practice). Exclude folds, edges, necrotic regions and poorly preserved areas using rules set before scoring (standard IHC practice). Record collection time when comparing biological groups because CRY1 is a core circadian-clock component (UniProt Q16526 function).

How do I distinguish convincing CRY1 staining from artefact?

A convincing pattern follows intact cells and resolves into nuclear or cytoplasmic signal, both reported locations for CRY1 (UniProt Q16526 subcellular location). Check whether staining tracks an identified cell population; HPA reports broad tissue expression but notes low consistency between antibody staining and RNA expression (HPA tissue IHC). Strong signal confined to section edges, folds, necrosis or extracellular material warrants repeat inspection with morphology and a no-primary control (standard IHC practice). Brown deposits that persist without primary antibody can reflect detection background, including endogenous enzyme activity in a chromogenic workflow (standard IHC practice). Interpret between-sample intensity cautiously unless tissue processing, scoring compartment and collection time are comparable (standard IHC practice; UniProt Q16526 function).

Boster reagents

Best CRY1 / Cryptochrome-1 IHC Antibodies

The IHC-validated antibody PB9540 has paraffin-section images from rat and mouse intestine (IHC image captions). Human, mouse and rat reactivity is listed; no IF data are supplied (catalog).

Real IHC data

Anti-Cryptochrome I/CRY1 Antibody ®

Cat # PB9540

View product →

PB9540 is the only SKU shown and lists IHC as an application for human, mouse and rat (catalog: applications and reactivity). Its IHC images show staining in paraffin sections of rat and mouse intestine (IHC image captions).

Which to pick: Choose PB9540 for paraffin-section tissue IHC: its own caption documents citrate retrieval at pH 6 for 20 minutes and staining at 1 μg/ml (PB9540 IHC image caption); the fixative is unreported (PB9540 IHC image caption). There is no IF/ICC-validated SKU to choose from this payload (catalog: applications and IF images). For cross-species planning, PB9540 lists human, mouse and rat reactivity and has a rabbit host, while clonality is unreported (catalog: reactivity, host and clone); the supplied IHC images document mouse and rat tissue only (IHC image 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.