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CYP27A1 / Sterol 26-hydroxylase, mitochondrial · IHC design guide
Design Immunohistochemistry for CYP27A1
Plan chromogenic IHC for CYP27A1 using the reported cytoplasmic tissue pattern and high staining in lung macrophages (HPA tissue IHC). The guide covers FFPE handling and interpretation alongside its mitochondrial inner membrane location (UniProt).
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); inner mitochondrial membrane is the molecular location (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues, including 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02121-2) | |
| Caveat | Lung macrophages can contribute endogenous peroxidase signal (HPA tissue IHC; standard IHC practice) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms; the mature chain starts at residue 34, so epitope position matters (UniProt) |
Section 1
Recommended CYP27A1 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A02121-2). The two published protocols below describe adrenal tissue and liver sections (PMC4618091; PMC8399082).
| 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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A02121-2). Both published protocols used citrate retrieval for CYP27A1 (PMC4618091; PMC8399082).
Section 2
What Is the Expected CYP27A1 Staining Pattern?
CYP27A1 associates with the mitochondrial inner membrane after import and has no transmembrane segment (UniProt Q02318 topology). In paraffin-section IHC, expect cytoplasmic staining, especially in HPA-high glandular cells, respiratory epithelial cells, kidney tubule cells, and lung macrophages (HPA tissue IHC). HPA rates tissue staining reliability Enhanced, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct cytoplasmic staining in kidney tubule cells or lung macrophages. | This matches HPA's High staining in those cell types (HPA tissue IHC). A granular cytoplasmic pattern is compatible with mitochondrial localisation, but chromogenic IHC cannot resolve the inner membrane (UniProt Q02318 topology; general IHC practice). |
| Predominantly nuclear or cell-surface staining, without a convincing cytoplasmic component. | That compartment conflicts with the reported mitochondrial inner-membrane association and HPA's cytoplasmic tissue profile (UniProt Q02318 topology; HPA tissue IHC). Check controls and morphology before interpreting it as CYP27A1. |
| Strong adipocyte, bone-marrow hematopoietic-cell, or ovarian stromal-cell staining. | HPA reports CYP27A1 as Not detected in those respective cell populations (HPA tissue IHC). Consider nonspecific antibody binding or endogenous chromogen-generating activity; compare with an antibody-omission control (general IHC practice). |
| Diffuse color across tissue, including spaces or structures without discernible cells. | A broad deposit does not establish the cell-specific cytoplasmic pattern reported by HPA (HPA tissue IHC). Background from detection chemistry or inadequate blocking is possible; inspect the antibody-omission control (general IHC practice). |
| No staining in kidney tubule cells when tissue morphology and counterstain are preserved. | HPA reports High staining in kidney tubule cells (HPA tissue IHC). Review the complete IHC run, including the validated antibody conditions and positive-control section, before calling the sample CYP27A1-negative (general IHC practice). |
💡Expected CYP27A1 appearanceA credible positive is discernible cytoplasmic staining in HPA-High cells, such as kidney tubule cells or lung macrophages; dominant nuclear or widespread cell-independent color is suspect (HPA tissue IHC; UniProt Q02318 topology; general IHC practice).
| Cell population and reference intensity | HPA reports High staining in adrenal gland, duodenum, seminal-vesicle and small-intestine glandular cells, respiratory epithelium of bronchus and nasopharynx, kidney tubule cells, and lung macrophages; its level applies to the named cells, not every cell in each tissue (HPA tissue IHC). |
| Subcellular interpretation | CYP27A1 is imported into mitochondria and associates with the inner membrane as a membrane-extrinsic protein, with no transmembrane segment (UniProt Q02318 topology). HPA describes mainly mitochondrial and additional cytosolic localisation by ICC-IF; either can contribute to cytoplasmic signal (HPA subcellular ICC-IF). |
| Antibody evidence | HPA059155 is listed as IHC Enhanced and ICC Approved (HPA antibodies). Enhanced supports the reported IHC pattern through independent-antibody reproduction or orthogonal data; it does not make every positive deposit specific (HPA antibodies; general IHC practice). |
| Low and undetected populations | HPA lists urothelial cells, prostate glandular cells, smooth muscle cells, skeletal myocytes, splenic red-pulp cells, and lymph-node germinal-center cells as Low; adipocytes, bone-marrow hematopoietic cells, and ovarian stromal cells are Not detected (HPA tissue IHC). Use these distinctions when judging contrast. |
| IF/ICC Q&A: what localisation should I expect? | Mainly mitochondrial signal with additional cytosolic signal is reported in ICC-IF (HPA subcellular ICC-IF). This describes the expected compartment; IF/ICC experimental conditions belong to its own guide (HPA subcellular ICC-IF). |
| Processing and epitope assumptions | UniProt annotates a mature chain at residues 34–531 and no annotated glycosylation sites or isoforms (UniProt Q02318 processing). The supplied sources do not locate the antibody epitope or establish CYP27A1-specific fixation or retrieval sensitivity; choose retrieval using the antibody's validated IHC-P instructions (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-control section has no discernible cytoplasmic stain. | The run may have failed, or the selected antibody conditions may be unsuitable; HPA reports High staining in kidney tubule cells and lung macrophages (HPA tissue IHC; general IHC practice). | Check detection reagents and the antibody's validated IHC-P conditions, then repeat with a documented HPA-High cell population in the control section (HPA tissue IHC; general IHC practice). |
| The antibody-omission control develops the same brown deposit as the stained section. | Endogenous chromogen-generating activity or detection-system background is possible (general IHC practice). | Review endogenous-enzyme blocking and detection reagents for the chromogen system; interpret sample signal only after the omission control is clean (general IHC practice). |
| Strong staining appears throughout adipocytes or bone-marrow hematopoietic cells. | Those populations are Not detected in HPA tissue IHC, so nonspecific binding or detection background is plausible (HPA tissue IHC; general IHC practice). | Compare omission and positive controls, assess cell morphology, and repeat using the antibody's validated IHC-P conditions before assigning CYP27A1 positivity (general IHC practice). |
| Only nuclei stain in a section with otherwise intact morphology. | Nuclear-only staining conflicts with mitochondrial association and the HPA cytoplasmic tissue profile (UniProt Q02318 topology; HPA tissue IHC). | Treat the nuclear pattern as unconfirmed; inspect controls and compare with cytoplasmic staining in an HPA-High cell population (HPA tissue IHC; general IHC practice). |
| A low-staining population looks negative beside a strongly positive control. | HPA rates urothelial cells and skeletal myocytes Low, whereas kidney tubule cells are High (HPA tissue IHC). A faint signal may be harder to distinguish from background (general IHC practice). | Judge each named cell population against its HPA level and the run's background controls; avoid using a Low population alone to declare assay failure (HPA tissue IHC; general IHC practice). |
| Cytoplasmic color is uniform and fine mitochondrial detail is not visible. | Chromogenic paraffin-section IHC has limited subcellular resolution; HPA calls tissue staining cytoplasmic, while ICC-IF reports mainly mitochondrial plus cytosolic localisation (HPA tissue IHC; HPA subcellular ICC-IF; general IHC practice). | Score the named cell population and cytoplasmic distribution against controls; do not infer inner-membrane localisation from chromogenic detail alone (UniProt Q02318 topology; general 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
🧪Start with lung, where macrophages should stain (High; HPA: lung macrophages), and run adipose tissue as a negative, where adipocytes are Not detected (HPA: adipose adipocytes). On the lung slide, cells without macrophage staining should remain at background; use them as an internal reference without assuming every other cell type is CYP27A1-negative (HPA: lung macrophages).
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 section and a matched rabbit IgG isotype control, alongside CYP27A1 knockout tissue or a validated peptide-block control (selected A02121-2 tissue-IHC caption: rabbit primary; standard IHC practice). Block endogenous peroxidase and check for pigment-related background in lung macrophages before interpreting DAB staining (HPA: 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 A02121-2 tissue-IHC caption). That example used heat-mediated retrieval in EDTA at pH 8.0, but retrieval dependence and whether frozen sections or IF are easier are unreported (selected A02121-2 tissue-IHC caption). For lung macrophages, endogenous peroxidase and pigment may complicate chromogenic scoring (HPA: lung macrophages; standard IHC practice).
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 sections by checking retrieval, cellular distribution, controls and scoring against the reported IHC image and reference expression patterns.
Which retrieval conditions should I try first for weak CYP27A1 staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A02121-2). The reported human liver IHC image used that retrieval, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A02121-2). If staining is weak, first check section handling, retrieval heating and primary incubation against the reported conditions, then compare a modified retrieval condition on adjacent sections with the same detection reagents (standard IHC practice). Keep a no-primary control and a tissue control in each comparison so stronger background is not mistaken for improved CYP27A1 detection (standard IHC practice).
How should I assess fixation when CYP27A1 staining varies between paraffin blocks?
CYP27A1-specific sensitivity to fixation is unknown from the supplied evidence; the reported paraffin-section image does not state its fixative (datasheet A02121-2). Record each block’s fixative, fixation duration and processing history before comparing signal, because these variables can change antigen accessibility in IHC (standard IHC practice). Process adjacent sections from the blocks together using EDTA retrieval at pH 8.0, the reported starting condition, and hold antibody and detection settings constant (datasheet A02121-2; standard IHC practice). If results differ, examine morphology and compare staining with a control block processed in the same run; do not assign the difference to a CYP27A1-specific fixation effect without further evidence (standard IHC practice).
What staining pattern is plausible for CYP27A1 in chromogenic sections?
Expect cellular staining compatible with a mitochondrial protein: CYP27A1 associates with the mitochondrial inner membrane after import and has no transmembrane segment (UniProt Q02318 localisation and topology). At tissue-IHC resolution, a granular or broadly cytoplasmic DAB pattern can be compatible with that location; IHC alone cannot resolve the inner membrane (UniProt Q02318 localisation; standard IHC practice). HPA reports cytoplasmic expression in most tissues and high staining in lung macrophages and kidney tubular cells, which can help select fields for review (HPA tissue IHC). Diffuse nuclear or extracellular staining warrants comparison with the no-primary control and tissue morphology before it is scored as CYP27A1 (UniProt Q02318 localisation; standard IHC practice).
Could epitope placement explain discrepant CYP27A1 staining?
The supplied record lists 0 isoforms and a processed chain spanning residues 34–531; it does not identify this antibody’s epitope (UniProt Q02318 processing and isoforms; datasheet A02121-2). Consequently, do not attribute differing IHC patterns to an alternative isoform or assume the antibody recognizes the mature chain without epitope documentation (UniProt Q02318 processing and isoforms; standard IHC practice). CYP27A1 has no annotated transmembrane segment and has reported acetyllysines at residues 283, 509 and 520, but their effects on this antibody’s staining are unknown (UniProt Q02318 topology and modified residues). Compare retrieval conditions and independently validated reagents on adjacent sections before making an epitope-based explanation (standard IHC practice).
How can I use IF to investigate an ambiguous CYP27A1 IHC pattern?
Use IF as a complementary localisation check: HPA reports mainly mitochondrial CYP27A1, with an additional cytosolic location, in its subcellular assessment (HPA subcellular). Multiplex CYP27A1 with a validated marker for the expected cell population, such as macrophages in lung, while keeping single-stain controls to assess channel bleed-through (HPA tissue IHC: High in lung macrophages; standard IF practice). Select fluorophores and imaging channels after measuring the specimen’s autofluorescence, and compare the CYP27A1 channel with secondary-only controls (standard IF practice). Because the antibody epitope is unspecified and CYP27A1 is imported into mitochondria, choose permeabilisation only after establishing whether the recognized epitope is accessible in the chosen preparation (datasheet A02121-2; UniProt Q02318 localisation; standard IF practice).
How do I distinguish CYP27A1 signal from chromogenic background?
Run a no-primary control through secondary antibody and DAB development to identify detection-related staining in the same tissue (standard IHC practice). The reported paraffin-section workflow used a peroxidase-conjugated secondary and DAB; include a peroxidase-blocking step as a general chromogenic IHC precaution and assess whether it reduces background (datasheet A02121-2; standard IHC practice). If diffuse color persists, review section edges, folds and damaged areas separately from intact cells, then compare blocking, washing and primary-antibody concentration on adjacent sections (standard IHC practice). The reported starting concentration is 2 μg/ml after 10% goat serum blocking; any adjustment needs matched controls and identical development time (datasheet A02121-2; standard IHC practice).
How should I score CYP27A1 staining across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and anatomical region before scoring, then exclude folds, necrosis and section edges using the same rules for every specimen (standard IHC practice). For cellular staining, record the percentage of positive cells and intensity categories, or calculate an H-score from those categories; keep the threshold anchored to controls processed in the same run (standard IHC practice). If cell abundance differs substantially, report positive cells per mm² and normalize that density to the evaluated viable area or the number of eligible cells (standard IHC practice). Report macrophages and epithelial or glandular cells separately where present, because HPA describes high CYP27A1 staining in distinct cell populations across tissues (HPA tissue IHC).
When should an apparent CYP27A1-positive cell be considered an artefact?
Judge signal against both compartment and cell identity: CYP27A1 is primarily mitochondrial, while HPA also reports a cytosolic location and cytoplasmic tissue staining (UniProt Q02318 localisation; HPA subcellular; HPA tissue IHC). High staining in lung macrophages and kidney tubular cells supports checking those populations, but does not make every brown structure within them specific (HPA tissue IHC; standard IHC practice). Treat staining confined to section edges, necrotic regions or a no-primary control as suspect, and investigate endogenous peroxidase when DAB signal persists without primary antibody (standard IHC practice). Confirm a disputed pattern with intact-cell morphology, matched controls and, when available, an independently validated localisation method before assigning it to CYP27A1 (standard IHC practice).
Boster reagents
Best CYP27A1 / Sterol 26-hydroxylase, mitochondrial IHC Antibodies
The IHC-validated antibody has illustrated staining in human paraffin-embedded liver sections and IF staining in A431 cells (A02121-2 image captions); its listed reactivity covers human, mouse, rat and monkey (A02121-2 catalog).
Real IHC data
Anti-CYP27A1 Antibody ®
Cat # A02121-2
A02121-2 has illustrated IHC staining in human paraffin-embedded liver sections and IF staining in A431 cells (A02121-2 image captions). M02121-1 lists IHC and IF/ICC applications and human, mouse and rat reactivity, but has no supplied IHC or IF image (M02121-1 catalog).
Which to pick: For tissue IHC, choose A02121-2: its own image shows staining in a human paraffin-embedded liver section after EDTA pH 8.0 retrieval, using 2 μg/ml primary antibody; the fixative is unreported (A02121-2 IHC image caption). For IF/ICC, A02121-2 also has an A431 cell image at 5 μg/ml, while M02121-1 is a rabbit monoclonal option with IF/ICC listed but no supplied IF image (A02121-2 IF image caption; M02121-1 catalog). For cross-species selection, A02121-2 lists human, mouse, rat and monkey reactivity; M02121-1 lists human, mouse and rat, with no supplied species-specific IHC images (A02121-2 catalog; M02121-1 catalog).
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); inner mitochondrial membrane is the molecular location (UniProt). 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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02121-2); Caveat: Lung macrophages can contribute endogenous peroxidase signal (HPA tissue IHC; standard IHC practice); Isoform / epitope: No annotated isoforms; the mature chain starts at residue 34, so epitope position matters (UniProt); Regulation: Expression regulation unreported (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.