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CYP27A1 / Sterol 26-hydroxylase, mitochondrial · IHC design guide
Design Immunohistochemistry for CYP27A1
Plan CYP27A1 staining in paraffin sections using the catalog antibody’s IHC protocol. Compare the observed cytoplasmic pattern with tissue IHC evidence, including high staining in lung macrophages (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYP27A1 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in most tissues; high in lung macrophages (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02121-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation not specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; mature chain spans residues 34–531 (UniProt) |
Section 1
Recommended CYP27A1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with three published CYP27A1 chromogenic IHC protocols (datasheet A02121-2; PMC4618091; PMC8399082; PMC10494299).
| Sample | Paraffin-embedded human liver tissue; fixative not specified (datasheet A02121-2) |
| Fixation | Image fixative and duration unreported (datasheet A02121-2); 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 A02121-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02121-2) |
| Primary antibody | Rabbit anti-CYP27A1, 2-5μg/ml (datasheet A02121-2) |
| Primary incubation | Overnight at 4 °C (datasheet A02121-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02121-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CYP27A1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A02121-2). Published CYP27A1 protocols also used heated citrate retrieval (PMC4618091; PMC8399082).
Section 2
What Is the Expected CYP27A1 Staining Pattern?
CYP27A1 staining in paraffin sections should be predominantly cytoplasmic, with strong signal in cell populations such as lung macrophages and kidney tubule cells (HPA: High in both; cytoplasmic expression in most tissues). The protein associates with the mitochondrial inner membrane after import and has no transmembrane segment (UniProt Q02318 topology). HPA rates tissue IHC reliability Enhanced, while reporting medium consistency between staining and RNA data (HPA tissue IHC).
| Strong cytoplasmic staining in lung macrophages or kidney tubule cells, with tissue structures still clear (HPA: High in both). | This fits the reported cell distribution and is a useful positive pattern for IHC interpretation (HPA tissue IHC). Chromogenic IHC can show cytoplasmic staining without resolving individual mitochondria (standard IHC practice). |
| Staining is mainly nuclear, or restricted to cell surfaces rather than cytoplasm. | That pattern conflicts with the reported cytoplasmic tissue profile and mitochondrial location (HPA tissue IHC; HPA subcellular; UniProt Q02318). Check staining controls and localization before assigning it to CYP27A1 (standard IHC practice). |
| Adipocytes, marrow hematopoietic cells, or ovarian stroma stain strongly (HPA: Not detected in these cells). | The result conflicts with the listed HPA tissue IHC observations; nonspecific antibody binding or endogenous chromogen activity are possibilities, not diagnoses (HPA tissue IHC; standard IHC practice). Review controls and cell identity. |
| Color spreads across tissue structures, including areas outside the expected positive cells. | Diffuse background prevents a reliable cell-specific call (standard IHC practice). Evaluate the negative detection control, blocking, washing, and development conditions before interpreting the distribution (standard IHC practice). |
| No signal appears in lung macrophages or kidney tubule cells (HPA: High in both). | A failed positive control is possible, but an HPA High designation does not guarantee every section will stain (HPA tissue IHC; standard IHC practice). Verify tissue identity and assay performance before calling the sample negative. |
💡Expected CYP27A1 appearanceCall a convincing positive result when cytoplasmic staining is strong in the specified lung macrophages or kidney tubule cells (HPA: High in both; cytoplasmic profile); predominant nuclear staining or strong signal in listed HPA-undetected cells is discordant (HPA tissue IHC; HPA subcellular).
| Compartment and optical resolution | CYP27A1 associates with the mitochondrial inner membrane as a membrane-extrinsic protein and has no transmembrane segment (UniProt Q02318); assess chromogenic sections for cytoplasmic distribution rather than requiring visible mitochondrial puncta (standard IHC practice). |
| Choice of positive and low-signal comparison cells | Lung macrophages and kidney tubule cells are reported High, while urothelial cells and skeletal myocytes are Low (HPA tissue IHC). Compare the named cell populations; a tissue label alone does not specify which cells should stain. |
| Interpretive confidence | The listed antibody HPA059155 has Enhanced IHC validation, but HPA describes only medium consistency between antibody staining and RNA expression (HPA antibodies; HPA tissue IHC). Treat a discordant pattern as requiring confirmation rather than assuming its identity. |
| Processing and epitope assumptions | UniProt lists a mature chain at residues 34–531 and three acetylated lysines, but no antibody epitope is supplied (UniProt Q02318). These annotations cannot predict which retrieval condition will work; target-specific fixation sensitivity is unreported in the supplied sources. |
| IF/ICC Q&A: what location should an image show? | Mainly mitochondrial signal, with additional cytosolic signal, is approved in HPA ICC-IF images (HPA subcellular). This localization helps interpret an image; it does not specify an IF/ICC preparation or detection protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal in an HPA High cell population (HPA tissue IHC). | The positive control or detection run may have failed; the supplied sources give no CYP27A1-specific fixation or retrieval effect (standard IHC practice; HPA tissue IHC). | Confirm the sampled cells, run an established positive section, and review the antibody's IHC-P instructions and detection controls (standard IHC practice). |
| Weak signal appears in cells listed as HPA High (HPA tissue IHC). | Underdevelopment, an unsuitable working dilution, or incomplete assay optimization can reduce visible chromogen (standard IHC practice); no dilution is supplied here. | Check control performance and optimize detection or dilution within the catalog antibody's IHC-P instructions (standard IHC practice); do not infer a CYP27A1-specific retrieval defect. |
| Nuclei dominate the staining while cytoplasm is faint. | The distribution disagrees with HPA's cytoplasmic tissue profile and approved mitochondrial ICC-IF location (HPA tissue IHC; HPA subcellular). | Inspect the negative detection control and compare a known positive cell population; repeat with validated IHC conditions if the mismatch persists (standard IHC practice). |
| Strong color appears in adipocytes or marrow hematopoietic cells (HPA: Not detected). | Nonspecific binding or endogenous detection activity may account for unexpected color (standard IHC practice); the observation alone cannot distinguish them. | Check the no-primary detection control and antibody blocking or dilution, then compare the named cells with a positive control section (standard IHC practice). |
| Background obscures cell boundaries across the section. | Insufficient washing, blocking, or excessive development can produce widespread background in chromogenic IHC (standard IHC practice). | Review the negative control and adjust washing, blocking, and chromogen development using routine IHC optimization (standard IHC practice); score only interpretable cells. |
| A low-signal cell population is scored negative after a faint stain. | HPA lists urothelial cells and skeletal myocytes as Low, which is distinct from Not detected (HPA tissue IHC). | Keep Low and Not detected categories separate; score the identified cell type against controls under the same detection conditions (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing CYP27A1 staining
A02121-2 · IHCIHC analysis of CYP27A1 using anti-CYP27A1 antibody (A02121-2). CYP27A1 was detected in a paraffin-embedded section of human liver tissue. H
A02121-2 · IFIF analysis of CYP27A1 using anti-CYP27A1 antibody (A02121-2). CYP27A1 was detected in immunocytochemical section of A431 cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135929-CYP27A1/tissue/cerebral+cortex#imid_17477887
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135929-CYP27A1/tissue/colon#imid_17477821
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135929-CYP27A1/tissue/liver#imid_17477806
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135929-CYP27A1/tissue/kidney#imid_17477807
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA059155; green = CYP27A1, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA059155; green = CYP27A1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYP27A1 IHC & IF
🧪Run lung first and require staining in macrophages (HPA: High in lung macrophages); use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes). On the positive slide, unstained neighboring cells should show only background signal; do not presume every nonmacrophage cell is negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYP27A1 in Hep-G2, SK-MEL-30, U2OS, Sperm, KOLF2.1J, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, nonimmune rabbit IgG matched to the primary antibody, and CYP27A1-knockout tissue or cells as a biological negative (selected caption: rabbit primary; standard IHC controls). For lung DAB staining, block endogenous peroxidase and distinguish macrophage pigment from chromogen using the controls and tissue morphology (HPA: High in lung macrophages; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02121-2 paraffin-section caption does not state a fixative (selected caption: fixative not stated). The caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is essential (selected caption: EDTA retrieval); lung macrophage pigment can complicate DAB interpretation (HPA: High in lung macrophages; standard IHC practice). For IF/ICC, mitochondrial localization has been observed, but the supplied evidence does not establish that IF/ICC or frozen sections are easier than paraffin IHC (HPA: approved mitochondrial ICC-IF localization).
HPA tissue IHC evidence for CYP27A1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
Section 3
Advanced CYP27A1 IHC Tips
Troubleshoot CYP27A1 staining in paraffin section chromogenic IHC using the catalog antibody’s tissue image, protein annotation, and tissue expression data.
Which retrieval conditions should I try first for CYP27A1 IHC?
Start with heat mediated retrieval in EDTA at pH 8.0 for CYP27A1 paraffin section IHC (datasheet A02121-2). The catalog antibody’s human liver image used this retrieval before staining with 2 μg/ml primary antibody overnight at 4°C (datasheet A02121-2). If staining is weak, adjust heating duration within the retrieval system’s instructions while keeping buffer and pH fixed, then compare sections processed together (standard IHC practice). Check that tissue remains intact and that staining appears in expected cellular regions; stronger signal alone does not establish specificity (standard IHC practice; HPA: cytoplasmic expression in most tissues).
Could fixation explain weak or uneven CYP27A1 staining?
The selected image documents a paraffin embedded human liver section but does not report its fixative, so CYP27A1 specific fixation sensitivity is unknown (datasheet A02121-2: fixative not stated). Record the fixative, fixation duration, section thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). If those conditions vary, stain matched sections in one run using the same EDTA pH 8.0 retrieval and 2 μg/ml primary antibody conditions (datasheet A02121-2; standard IHC practice). Treat any association between fixation history and signal as an experimental observation requiring matched controls, rather than an established property of CYP27A1 (standard IHC practice).
What staining pattern fits CYP27A1 in paraffin sections?
Expect predominantly cytoplasmic staining at light microscope resolution, with a pattern compatible with mitochondrial enrichment (HPA: cytoplasmic expression in most tissues; UniProt Q02318: mitochondrial inner membrane association). CYP27A1 is imported into mitochondria and then associates with the inner membrane as a membrane extrinsic protein; it has no annotated transmembrane segment (UniProt Q02318 topology). HPA also reports an approved cytosolic location, so diffuse cytoplasmic signal does not by itself exclude genuine expression (HPA subcellular: mitochondria and cytosol). Compare staining with a matched negative control and assess nuclear only or extracellular staining cautiously, especially if it also appears in the control (standard IHC practice).
How should epitope uncertainty affect CYP27A1 IHC interpretation?
The record lists 0 alternative isoforms and identifies the processed chain as residues 34–531, but no antibody epitope is supplied here (UniProt Q02318 processing and isoforms; datasheet A02121-2: epitope not supplied). Consequently, neither mature chain recognition nor sensitivity to precursor processing can be assigned to this antibody from these data (UniProt Q02318 processing; datasheet A02121-2: epitope not supplied). Acetylated residues are annotated at 283, 509, and 520; their effect on this antibody’s IHC staining is unknown (UniProt Q02318 modified residues; datasheet A02121-2: epitope not supplied). When an unexpected pattern persists, seek epitope information and compare independent staining controls before attributing it to protein processing (standard IHC practice).
How can IF help assess a CYP27A1 IHC pattern?
For a separate IF experiment, multiplex CYP27A1 with a marker of the expected cell population, such as a macrophage marker when investigating lung macrophages (HPA tissue IHC: high in lung macrophages; standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence and include single stain controls for bleed through (standard IF practice). CYP27A1 reaches the mitochondrial matrix and associates with the inner membrane without an annotated transmembrane segment, so permeabilisation must allow antibody access to the epitope’s compartment (UniProt Q02318 topology; standard IF practice). Because this payload gives no antibody epitope or IF fixation condition, optimise membrane access and fixation empirically with appropriate controls (datasheet A02121-2: epitope not supplied; standard IF practice).
How do I reduce diffuse brown background without losing CYP27A1 signal?
Begin by comparing the stained section with a primary antibody omission control, then check whether background tracks tissue edges, folds, or damaged areas (standard IHC practice). The catalog image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase based DAB detection (datasheet A02121-2). If diffuse staining persists, verify the peroxidase block, adjust washing or antibody concentration systematically, and keep DAB development consistent across sections (standard chromogenic IHC practice). Evaluate any reduction against expected cytoplasmic staining rather than treating a uniformly pale section as an improvement (HPA: cytoplasmic expression in most tissues; standard IHC practice).
How should CYP27A1 IHC be scored across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cellular compartment and cell population before scoring, because HPA reports cytoplasmic staining across many tissues but differing levels among cell types (HPA tissue IHC profile and positive/negative listings). For a defined population, record the percentage of positive cells and staining intensity, or calculate an H-score using consistent intensity categories (standard IHC quantification practice). If the question concerns infiltrating positive cells, report their density per mm² of evaluable tissue and identify the counted cell population (standard IHC quantification practice). Normalise comparisons to the same tissue area or eligible cell count, exclude damaged regions consistently, and use fixed imaging and scoring settings (standard IHC quantification practice).
What separates convincing CYP27A1 staining from artefact?
A convincing result places signal in plausible cells and a predominantly cytoplasmic compartment, consistent with HPA tissue staining and mitochondrial localisation (HPA tissue IHC profile; UniProt Q02318 subcellular location). High staining in lung macrophages or kidney tubular cells is supported by HPA, whereas adipocytes are listed as not detected and warrant closer scrutiny if strongly positive (HPA tissue IHC: lung, kidney, and adipose tissue). Nuclear only, extracellular, edge restricted, or necrotic staining should be checked against morphology and controls before interpretation (standard IHC practice). Rule out endogenous peroxidase contribution with an appropriate control, and confirm that any DAB signal depends on the primary antibody (standard chromogenic IHC practice).
Boster reagents
Best CYP27A1 / Sterol 26-hydroxylase, mitochondrial IHC Antibodies
A02121-2 has IHC data from human liver paraffin sections and IF data from A431 cells; listed reactivity includes human, mouse, rat and monkey (A02121-2 image captions; catalog reactivity).
Real IHC data
Anti-CYP27A1 Antibody ®
Cat # A02121-2
The sole rendered card is A02121-2, whose IHC image shows human liver paraffin sections (cards payload; A02121-2 IHC caption). Its IF image shows A431 cells; M02121-1 has no supplied IHC or IF image (A02121-2 IF caption; M02121-1 image fields).
Which to pick: Choose A02121-2 for tissue IHC using the documented human liver paraffin-section example; the fixative is unreported (A02121-2 IHC caption). Choose A02121-2 for IF/ICC when an image-backed example is useful, based on its A431-cell IF image (A02121-2 IF caption). For cross-species work, A02121-2 lists human, mouse, rat and monkey reactivity, while M02121-1 is a rabbit monoclonal listing human, mouse and rat; the supplied IHC image documents human tissue only (catalog reactivity; M02121-1 catalog entry; A02121-2 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q02318 (CP27A_HUMAN, Sterol 26-hydroxylase, mitochondrial).
- Human Protein Atlas. CYP27A1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CYP27A1 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the cytosol..
- Human Protein Atlas. CYP27A1 antibody validation summary (1 antibodies).
- Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway. Circulation. Cardiovascular genetics 2015 — PMC4618091.
- Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of cerebrotendinous xanthomatosis. Molecular therapy. Methods & clinical development 2021 — PMC8399082.
- Tissue specific role of ABCA1 in lung cholesterol homeostasis under high-cholesterol diet. Frontiers in nutrition 2025 — PMC12343695.
- 27-Hydroxycholesterol represses G9a expression via oestrogen receptor alpha in breast cancer. Journal of cellular and molecular medicine 2023 — PMC10494299.
- PubMed PMID:1708392 — UniProt-cited evidence.
- PubMed PMID:7690968 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYP27A1 (Sterol 26-hydroxylase, mitochondrial). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02121-2). Validated IHC antibody: A02121-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver tissue; fixative not specified (datasheet A02121-2); 2. Fixation: Image fixative and duration unreported (datasheet A02121-2); 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 A02121-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02121-2); 8. Primary antibody: Rabbit anti-CYP27A1, 2-5μg/ml (datasheet A02121-2); 9. Primary incubation: Overnight at 4 °C (datasheet A02121-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain spans residues 34–531 (UniProt); Regulation: Expression regulation not specified (UniProt). Evidence: UniProt Q02318, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A02121-2, PMC4618091, PMC8399082. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYP27A1/immunohistochemistry-ihc-guide.