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CYP2R1 / Vitamin D 25-hydroxylase · IHC design guide
Design Immunohistochemistry for CYP2R1
Plan CYP2R1 paraffin IHC with the catalog antibody at 1:100–1:300 (datasheet). Use adrenal gland glandular cells as a high staining reference and score the mainly cytoplasmic signal by cell type (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYP2R1 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 | Mainly cytoplasmic (HPA tissue IHC); ER membrane (UniProt) | |
| Staining pattern | Mainly cytoplasmic in glandular and other cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Medium staining–RNA concordance; verify positives (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms; signal peptide 1–26 is cleaved (UniProt) |
Section 1
Recommended CYP2R1 IHC & IF Protocols
The catalog antibody protocol is accompanied by published CYP2R1 IHC methods for ewe endometrium, human liver and colorectal tumor sections (PMC9835233; PMC8494743; PMC5216813).
| Sample | Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A03557) |
| Fixation | Image fixative and duration unreported (datasheet A03557); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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 anti-CYP2R1, 1:100-1:300 (datasheet A03557) |
| 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 | CYP2R1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval for the catalog antibody (page retrieval guidance); published CYP2R1 methods also include microwave citrate retrieval and staining without retrieval (PMC8494743; PMC5216813).
Section 2
What Is the Expected CYP2R1 Staining Pattern?
CYP2R1 is associated with the endoplasmic reticulum and microsome membranes, although no transmembrane segment is annotated (UniProt Q6VVX0 topology). In tissue sections, expect mainly cytoplasmic staining, including glandular, respiratory epithelial, and hematopoietic cells in the high-staining examples listed by HPA (HPA: tissue IHC). HPA rates the tissue pattern Approved, with medium consistency between staining and RNA data and external verification pending (HPA: reliability).
| Cytoplasmic staining in adrenal gland or breast glandular cells, bronchial respiratory epithelial cells, or bone marrow hematopoietic cells (HPA: High). | This matches the main tissue pattern (HPA: mainly cytoplasmic expression). Cytoplasmic chromogen is compatible with an ER- and microsome-associated protein; routine IHC does not resolve the exact membrane carrying the signal (UniProt Q6VVX0 location; general IHC practice). |
| Predominantly nuclear, sharply cell-surface, or extracellular staining, with little cytoplasmic signal. | These compartments conflict with the reported cytoplasmic tissue pattern and ER/microsome location (HPA: tissue IHC; UniProt Q6VVX0 location). Treat the result as suspect and check whether it persists with an appropriate detection control (general IHC practice). |
| Strong staining in a cell population outside the cited high-staining examples, without the expected pattern in those examples. | Possible explanations include cross-reactivity or endogenous detection activity (general IHC practice). The HPA examples are not an exclusive cell-type list: expression is mainly cytoplasmic in most tissues, and RNA tissue specificity is low (HPA: tissue IHC). |
| Diffuse chromogen across tissue and surrounding areas, obscuring cell boundaries. | This is difficult to score as CYP2R1 because HPA describes a cellular, mainly cytoplasmic pattern (HPA: tissue IHC). Review the secondary-only control, blocking, washes, and detection development for background (general IHC practice). |
| No visible signal in a section expected to contain adrenal glandular or bronchial respiratory epithelial cells. | Both are high-staining HPA examples, so a blank result warrants a workflow check (HPA: adrenal gland and bronchus, High). It does not alone prove CYP2R1 absence; HPA rates antibody–RNA consistency as medium, with external verification pending (HPA: reliability). |
💡Expected CYP2R1 appearanceCall a result consistent when staining is mainly cytoplasmic in the relevant glandular, respiratory epithelial, or hematopoietic cells, with conspicuous signal in HPA high-staining examples; dominant nuclear, extracellular, or diffuse background signal is suspect (HPA: tissue IHC; UniProt Q6VVX0 location).
| Tissue and cell selection | HPA lists high staining in adrenal, appendix, breast, duodenum, epididymis, and gallbladder glandular cells; bronchial respiratory epithelial cells; and bone marrow hematopoietic cells (HPA: tissue IHC). Lymph-node non-germinal-center cells are listed as Low, not negative (HPA: lymph node). |
| Strength of tissue evidence | The HPA tissue profile is Approved, but staining and RNA show medium consistency and external verification is pending (HPA: reliability). Antibody HPA042949 has Approved IHC status; no Enhanced IHC designation is supplied (HPA: antibody validation). Interpret an unexpected pattern cautiously. |
| Localization and processing | UniProt places CYP2R1 at ER and microsome membranes, annotates a signal sequence at residues 1–26 and a chain at 27–501, and annotates no transmembrane segment (UniProt Q6VVX0). Epitope position is unspecified, so these annotations cannot predict retrieval needs or staining loss. |
| IF/ICC applicability — Q&A | Can the tissue IHC pattern define an IF/ICC result? No: HPA provides no main subcellular IF location or ICC-IF images for CYP2R1 (HPA: subcellular). The ER/microsome annotation offers a localization expectation, but direct IF confirmation is unavailable here (UniProt Q6VVX0 location; HPA: subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| A high-staining reference tissue shows no chromogen (HPA: tissue IHC). | The section or detection workflow may have failed; a blank slide alone cannot establish absent protein (general IHC practice). | Confirm the expected cells are present, then check reagent order, positive control, retrieval procedure, and detection activity (general IHC practice). |
| The signal is mainly nuclear or outlines cell surfaces. | That distribution disagrees with mainly cytoplasmic tissue staining and ER/microsome localization (HPA: tissue IHC; UniProt Q6VVX0). | Compare with the negative detection control and repeat staining using the IHC-validated antibody under its documented conditions (general IHC practice). |
| The entire section develops diffuse color. | Nonspecific detection or excess chromogen development can obscure a cellular pattern (general IHC practice). | Inspect the secondary-only control; review blocking, washes, and development time before scoring cells (general IHC practice). |
| Unexpected cells stain strongly while cited high-staining cells do not. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA examples are not an exhaustive cell map (HPA: tissue IHC). | Check controls and repeat in a cited high-staining tissue. Record the cell identity and compartment before attributing signal to CYP2R1 (HPA: tissue IHC; general IHC practice). |
| Lymph-node non-germinal-center cells stain faintly. | Low staining is reported for this population (HPA: lymph node). Faint signal alone is therefore inconclusive. | Assess cellular localization and background alongside a high-staining reference tissue; do not use this population as a verified negative control (HPA: tissue IHC; general IHC practice). |
| An IF/ICC image appears inconsistent with the tissue IHC result. | HPA supplies no CYP2R1 ICC-IF images or main IF location for a direct comparison (HPA: subcellular). | Interpret the IF/ICC result within its own controls; use ER/microsome localization only as a qualified expectation (UniProt Q6VVX0 location; general IF practice). |
Experiment evidence
IHC & IF images showing CYP2R1 staining
A03557 · IHCImmunohistochemistry analysis of paraffin-embedded human colon carcinoma, using Cytochrome P450 2R1 Antibody. The picture on the right is bl
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000186104-CYP2R1/tissue/cerebral+cortex#imid_12041579
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000186104-CYP2R1/tissue/colon#imid_12038681
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000186104-CYP2R1/tissue/liver#imid_12038698
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000186104-CYP2R1/tissue/kidney#imid_12038683
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYP2R1 IHC & IF
🧪Run adrenal gland first and score its glandular cells, which HPA rates High for CYP2R1 (HPA: adrenal gland, glandular cells, High). HPA detects CYP2R1 in all 45 scored tissues, so no tissue is a validated negative; use no-primary and isotype controls, and treat unstained cells within the positive section as background comparators rather than proven negative cells (HPA: no negative tissue rows; detected in all 45 tissues).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CYP2R1 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 carries no ICC-IF cell line for CYP2R1; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only slide and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use a CYP2R1 knockout specimen, if available, as a biological negative, or compare with peptide-blocked staining as shown for SKU A03557 (standard IHC practice; selected-SKU tissue-IHC caption: peptide block). Check endogenous peroxidase background in the adrenal gland section before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption: fixative not stated). Antigen-retrieval dependency is also unreported; establish retrieval conditions empirically for the IHC-validated antibody (supplied target/application evidence; standard IHC practice). The evidence does not establish that frozen sections or IF are easier, and HPA lists no CYP2R1 ICC-IF image-bearing cell lines; assess adrenal gland background, including endogenous peroxidase, when scoring chromogenic staining (HPA: subcellular record; standard IHC practice).
HPA tissue IHC evidence for CYP2R1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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: CYP2R1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CYP2R1 IHC Tips
Troubleshoot CYP2R1 staining in paraffin sections using its ER localisation, the selected antibody image, and the limits of the available tissue evidence.
How should I retrieve CYP2R1 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Let sections cool in the retrieval buffer, then compare staining with a control section processed in the same run (standard IHC practice). If signal remains weak, test a second retrieval condition on adjacent sections while keeping antibody incubation and detection constant (standard IHC practice). Score whether glandular-cell cytoplasmic staining improves without a parallel rise in diffuse background; that pattern is consistent with the reported tissue profile and localisation (HPA: glandular-cell staining; HPA: mainly cytoplasmic expression).
Could fixation explain inconsistent CYP2R1 staining between paraffin blocks?
Target-specific sensitivity of CYP2R1 to fixation is unknown from the supplied evidence; the selected image identifies paraffin-embedded human colon carcinoma but does not state its fixative (A03557 tissue-IHC caption). Record each block’s fixative, fixation duration, and processing history before comparing staining intensity (standard IHC practice). Run blocks together with identical pH 6.0 retrieval for 20 min, antibody incubation, and chromogenic development (page retrieval setting; standard IHC practice). If staining differs, inspect morphology and a run control before attributing the difference to fixation; the caption and tissue profile do not establish a CYP2R1-specific fixation effect (A03557 tissue-IHC caption; HPA: tissue IHC profile).
What staining pattern fits CYP2R1, and how should I assess unexpected nuclear signal?
Assess CYP2R1 primarily as cytoplasmic staining in paraffin sections, because the tissue profile reports mainly cytoplasmic expression and the protein is assigned to ER and microsome membranes (HPA: mainly cytoplasmic expression; UniProt Q6VVX0 localisation). Its record lists no transmembrane segment, so do not treat a crisp cell-surface outline as the expected pattern (UniProt Q6VVX0 topology). For unexpected nuclear staining, compare adjacent sections stained with and without primary antibody and inspect whether signal follows damaged or folded tissue (standard IHC practice). Score the cytoplasmic compartment separately from nuclear or surface signal so an anomalous pattern cannot inflate the CYP2R1 result (standard IHC scoring practice).
How should I troubleshoot discordant results from CYP2R1 antibodies against different epitopes?
Map each stated immunogen or epitope against the 501-aa precursor and its annotated signal segment at residues 1–26 (UniProt Q6VVX0 processing). The annotated mature chain spans residues 27–501, while no alternative isoforms or glycosylation sites are listed in the supplied record (UniProt Q6VVX0 processing and annotations). Compare antibodies on adjacent sections under matched retrieval and detection conditions, scoring compartment and cell type as well as intensity (standard IHC practice). The A03557 image includes a synthesized-peptide blocking comparison, which supports assessment of that image but does not establish the epitope or validate another antibody (A03557 tissue-IHC caption).
How can I check a CYP2R1 IF result against the chromogenic IHC pattern?
Use an adjacent paraffin section to compare IF staining with cytoplasmic chromogenic staining, while treating IF as a separately optimised assay (HPA: mainly cytoplasmic expression; standard IF practice). Multiplex CYP2R1 with a validated marker for the expected glandular cells, and include single-label controls to check channel bleed-through (HPA: high staining in glandular cells of listed tissues; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence in unstained sections, favouring a channel with a clear signal-to-background difference (standard IF practice). Epitope sidedness is unreported, so test permeabilisation conditions appropriate to the antibody’s documented epitope location before interpreting absent intracellular signal (UniProt Q6VVX0 localisation; standard IF practice).
What should I change when diffuse brown background obscures CYP2R1 staining?
Check a section without primary antibody alongside the stained section to separate detection-system background from antibody-dependent staining (standard IHC practice). For peroxidase-based chromogenic detection, verify the peroxidase block, blocking step, washes, and DAB development time under matched conditions (standard IHC practice). If background remains diffuse, adjust antibody concentration and incubation on adjacent sections, recording the settings rather than assuming a target-specific dilution (standard IHC practice). Judge the result against mainly cytoplasmic staining and the available peptide-blocked A03557 image, while recognising that peptide competition alone does not prove every stained structure is CYP2R1 (HPA: tissue IHC profile; A03557 tissue-IHC caption).
How should I score CYP2R1 IHC across sections with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, using the reported glandular-cell staining as a guide where those cells are present (HPA: high staining in glandular cells of listed tissues; HPA: mainly cytoplasmic expression). Record the percentage of positive cells and intensity from 0–3, then calculate an H-score on the 0–300 scale (standard IHC scoring practice). Sample comparable viable areas and keep retrieval, imaging, and chromogen development consistent across sections (standard IHC practice). Normalise positive counts to the number of cells in the defined population, or report positive-cell density per mm² with the measured area stated (standard IHC quantification practice).
When should I doubt an apparent CYP2R1-positive region?
A credible result should be evaluated for cytoplasmic staining in intact cells, consistent with the tissue profile and ER or microsome assignment (HPA: mainly cytoplasmic expression; UniProt Q6VVX0 localisation). Treat predominantly nuclear or sharp surface staining as discordant with that expected pattern and investigate it with controls (HPA: tissue IHC profile; UniProt Q6VVX0 topology; standard IHC practice). Exclude edge staining, necrotic areas, and signal reproduced without primary antibody; check endogenous peroxidase when using peroxidase detection (standard IHC practice). Interpret a positive region cautiously because the tissue-IHC reliability is Approved with medium staining-to-RNA consistency and pending external verification (HPA: tissue IHC reliability).
Boster reagents
Best CYP2R1 / Vitamin D 25-hydroxylase IHC Antibodies
A03557 has a real IHC image from a human paraffin-embedded colon carcinoma sample (catalog IHC image caption). IF is listed for human, monkey and mouse (catalog applications; catalog reactivity).
Real IHC data
Anti-Vitamin D 25-hydroxylase CYP2R1 Antibody
Cat # A03557
A03557 has IHC image data from paraffin-embedded human colon carcinoma, including a peptide-blocked comparison (catalog IHC image caption). IF is listed as an application, with human, monkey and mouse reactivity; no IF image is supplied (catalog applications; catalog reactivity; catalog IF image alts).
Which to pick: For tissue IHC, choose A03557: its own image caption documents staining of a paraffin-embedded human colon carcinoma sample, but does not report the fixative (catalog IHC image caption). For IF, A03557 is the listed option; ICC use requires separate validation because ICC is not listed (catalog applications). For cross-species work, A03557 is listed as a rabbit polyclonal reactive with human, monkey and mouse (catalog host; catalog dilution_raw; catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6VVX0 (CP2R1_HUMAN, Vitamin D 25-hydroxylase).
- Human Protein Atlas. CYP2R1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CYP2R1 subcellular location (ICC-IF): Highest expression in NB4: 12.0 nTPM.
- Human Protein Atlas. CYP2R1 antibody validation summary (1 antibodies).
- Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes. Journal of animal science and biotechnology 2023 — PMC9835233.
- Effect and mechanism of vitamin D activation disorder on liver fibrosis in biliary atresia. Scientific reports 2021 — PMC8494743.
- Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer. Oncotarget 2016 — PMC5216813.
- Differential Expression of Prostaglandin I2 Synthase Associated with Arachidonic Acid Pathway in the Oral Squamous Cell Carcinoma. Journal of oncology 2018 — PMC6250001.
- PubMed PMID:12867411 — UniProt-cited evidence.
- PubMed PMID:15465040 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYP2R1 (Vitamin D 25-hydroxylase). Expected localisation Mainly cytoplasmic (HPA tissue IHC); ER membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03557 at 1:100-1:300 (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 workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A03557); 2. Fixation: Image fixative and duration unreported (datasheet A03557); 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 citrate buffer, pH 6.0 , 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 anti-CYP2R1, 1:100-1:300 (datasheet A03557); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Medium staining–RNA concordance; verify positives (HPA tissue IHC); Isoform / epitope: No isoforms; signal peptide 1–26 is cleaved (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt Q6VVX0, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in NB4: 12.0 nTPM, Datasheet A03557, PMC9835233, PMC8494743. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYP2R1/immunohistochemistry-ihc-guide.