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CYP51A1 / Lanosterol 14-alpha demethylase · IHC design guide
Design Immunohistochemistry for CYP51A1
Plan chromogenic CYP51A1 IHC-P around cytoplasmic staining in testicular Leydig cells (HPA tissue IHC). This guide covers formalin-fixed paraffin sections and the catalog antibody’s 1:10–1:50 starting dilution (datasheet: A04032-2 IHC-P).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYP51A1 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 | Leydig-cell cytoplasm (HPA tissue IHC); ER membrane (UniProt) | |
| Staining pattern | High cytoplasmic staining in testicular Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Use formalin-fixed paraffin sections (datasheet: A04032-2 IHC-P) (selected-SKU IHC image A04032-2); keep fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Antibody may detect protein from another gene (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended CYP51A1 IHC & IF Protocols
The catalog antibody protocol is followed by a published CYP51A1 IHC workflow for colorectal cancer (PMC5216813).
| Sample | FFPE human prostate carcinoma tissue (datasheet A04032-2) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A04032-2); 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-CYP51A1, 1:10-1:50 (datasheet A04032-2) |
| 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 | CYP51A1-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in Leydig cells. No signal in the no-primary control. |
💡Decision noteStart with citrate buffer at pH 6; the published CYP51A1 workflow used microwave retrieval for 20 min (PMC5216813).
Section 2
What Is the Expected CYP51A1 Staining Pattern?
CYP51A1 is an endoplasmic reticulum and microsome membrane protein with one transmembrane segment at residues 30–50 (UniProt Q16850 topology). In paraffin-section IHC, expect cytoplasmic staining in testicular Leydig cells, where HPA reports High staining (HPA: testis tissue IHC). HPA rates the tissue staining Approved but notes low agreement with RNA data and possible staining of proteins from more than one gene (HPA: tissue IHC reliability).
| Strong cytoplasmic staining in Leydig cells, with surrounding structures comparatively faint (HPA: High in Leydig cells). | This matches the reported positive cell type and compartment (HPA: testis tissue IHC). An ER-associated distribution is consistent with CYP51A1 topology (UniProt Q16850 topology); chromogenic IHC alone does not resolve the ER membrane. |
| Predominantly nuclear, surface, or extracellular staining in place of the expected cytoplasmic pattern (HPA: cytoplasmic in Leydig cells). | Treat this as a pattern mismatch, potentially caused by an artefact or nonspecific detection. Review the negative control and antibody conditions before assigning the signal to CYP51A1 (standard IHC practice). |
| Strong staining in adipocytes or bronchial respiratory epithelial cells (HPA: Not detected in these cells). | This conflicts with the reported cell-specific observations; cross-reactivity or endogenous detection activity is possible, not established. Check a no-primary control and compare staining with the reported Leydig-cell pattern (standard IHC practice; HPA: tissue IHC). |
| Diffuse colour across tissue and empty areas, obscuring cell boundaries (standard IHC practice). | This is background rather than an interpretable cell-specific pattern. Check blocking, antibody concentration, washing and the no-primary control before scoring cells (standard IHC practice). |
| No detectable staining in testicular Leydig cells (HPA: High in Leydig cells). | A negative result in the reported positive cell type calls for a run-level check. Confirm tissue identity, antibody performance and the IHC-P workflow before interpreting other negative cells (HPA: tissue IHC; standard IHC practice). |
💡Expected CYP51A1 appearanceCall a result positive when Leydig cells show High cytoplasmic staining (HPA: testis tissue IHC); isolated nuclear, surface, extracellular or diffuse background colour is a pattern mismatch (HPA: cytoplasmic profile; standard IHC practice).
| Membrane topology | CYP51A1 has one transmembrane segment at residues 30–50 and localises to ER and microsome membranes (UniProt Q16850 topology). Use that location to assess the staining pattern; the record does not locate the antibody epitope. |
| Tissue and cell choice | Leydig cells provide the reported High IHC example, while adrenal glandular cells are reported Low (HPA: tissue IHC). An adrenal result may therefore be harder to judge visually than the Leydig-cell example; compare identified cells, not whole-tissue averages. |
| Negative comparisons | HPA reports Not detected for specific cells, including adipocytes and bronchial respiratory epithelial cells (HPA: tissue IHC). These observations support cell-level comparison, but do not establish that every cell in those tissues is negative. |
| Antibody evidence and limits | Two rabbit polyclonal antibodies have Approved IHC status, HPA041325 and HPA043508 (HPA: antibody validation). HPA also flags low staining-to-RNA consistency, possible targeting of proteins from multiple genes and a splice or transcript discrepancy (HPA: tissue IHC reliability); interpret unexpected positives cautiously. |
| IF/ICC Q&A: should the same compartment appear? | Yes: HPA reports an Approved ER location by ICC-IF (HPA: subcellular ICC-IF). Its multi-gene antibody caution still applies (HPA: subcellular ICC-IF). Use the separate IF/ICC guide for that application; the IHC-P staining pattern here does not supply an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Leydig-cell signal is absent or very weak (HPA: High in Leydig cells). | The staining run or tissue assignment may need review; this result conflicts with the HPA positive example (HPA: testis tissue IHC). | Confirm Leydig-cell identity and review the catalog antibody’s IHC-P procedure, including its stated retrieval and dilution conditions; compare with a positive control (standard IHC practice). |
| Most structures show similar colour, including areas without the expected Leydig-cell pattern (HPA: cytoplasmic in Leydig cells). | Broad background can obscure a cell-specific chromogenic signal (standard IHC practice). | Inspect a no-primary control; review blocking, antibody concentration and washes using the antibody’s IHC-P procedure (standard IHC practice). |
| Adipocytes stain strongly despite the reported negative observation (HPA: Not detected in adipocytes). | Possible nonspecific binding or endogenous detection activity; the image alone cannot distinguish them (standard IHC practice). | Compare no-primary and relevant detection controls, then repeat with adjusted blocking or antibody concentration if controls indicate background (standard IHC practice). |
| Signal is mainly nuclear or at the cell surface rather than cytoplasmic (HPA: cytoplasmic in Leydig cells). | The compartment differs from the observed IHC pattern and expected ER membrane location (HPA: tissue IHC; UniProt Q16850 topology). | Recheck cell identification and controls; score the atypical signal separately while reviewing the antibody’s IHC-P conditions (standard IHC practice). |
| Two antibodies give different cell patterns in the same tissue (HPA: HPA041325 and HPA043508 IHC Approved). | Approved status does not remove HPA’s low RNA agreement, multi-gene targeting caution or splice/transcript discrepancy (HPA: tissue IHC reliability). | Document each antibody’s cell type and compartment separately, compare controls and avoid treating agreement in colour alone as CYP51A1 specificity (standard IHC practice). |
| A tissue section appears entirely negative although UniProt describes broad expression (UniProt Q16850 tissue specificity). | Broad expression does not guarantee detectable chromogenic staining in every cell; HPA reports Low adrenal glandular staining and several cell-specific negatives (HPA: tissue IHC). | Check the named cell population and run controls before scoring the section; use the reported Leydig-cell example as the positive pattern reference (HPA: testis tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing CYP51A1 staining
A04032-2 · IHCFormalin-fixed and paraffin-embedded human prostate carcinoma reacted with CYP51A1 Antibody (Center) , which was peroxidase-conjugated to th
A04032-1 · IFIF analysis of CYP51A1/CYP51 using anti-CYP51A1/CYP51 antibody (A04032-1). CYP51A1/CYP51 was detected in immunocytochemical section of CACO-
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000001630-CYP51A1/tissue/cerebral+cortex#imid_10684503
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000001630-CYP51A1/tissue/colon#imid_10684437
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000001630-CYP51A1/tissue/liver#imid_10684430
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000001630-CYP51A1/tissue/kidney#imid_10684423
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041325; green = CYP51A1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041325; green = CYP51A1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYP51A1 IHC & IF
🧪Run testis first and score Leydig cells, which show High CYP51A1 staining (HPA: High in testis Leydig cells). Use adipose tissue adipocytes as the biological low-expression comparator (HPA: Not detected in adipocytes); on the testis slide, assess non-Leydig cells for background staining without assuming they are CYP51A1-negative (HPA: Leydig cells High).
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYP51A1 in A-431, U-251MG, U2OS, MCF-7, with annotated localisation: Endoplasmic reticulum (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a control immunoglobulin matched to the primary antibody’s host species and, for a monoclonal antibody, isotype (standard IHC practice). Use CYP51A1 knockout material or a peptide-block control if the immunizing peptide is available, and quench endogenous peroxidase before chromogenic detection (standard IHC practice).
⚠️Feasibility: The selected antibody caption documents formalin-fixed, paraffin-embedded prostate carcinoma with peroxidase/DAB detection (catalog antibody A04032-2 caption); it reports no CYP51A1-specific fixation window. A target-specific antigen-retrieval requirement or effect is unreported in the supplied evidence, so optimize retrieval empirically (supplied evidence; standard IHC practice). The evidence does not establish that frozen sections or IF are easier; for IF, assess tissue autofluorescence alongside the no-primary control (standard IF practice).
HPA tissue IHC evidence for CYP51A1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Caution, Splice and/or transcript discrepancy exists.). 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CYP51A1 IHC Tips
Troubleshoot CYP51A1 chromogenic IHC by checking retrieval, cellular distribution and controls before comparing staining across paraffin sections.
How should I retrieve CYP51A1 in paraffin sections when staining is weak?
Use citrate buffer at pH 6.0 for heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic/membrane antigen). Let slides cool in buffer, then compare the same tissue with a matched section processed without retrieval to judge whether heating improves specific cytoplasmic staining (standard IHC practice; HPA tissue IHC: cytoplasmic expression in Leydig cells). Keep antibody concentration and DAB development time constant while assessing retrieval, so a darker result can be attributed to that step (standard IHC practice). If staining remains weak, vary retrieval time cautiously on serial sections and inspect tissue integrity before changing the antibody dilution (standard IHC practice).
Could fixation explain weak or uneven CYP51A1 staining?
Target-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence, so treat fixation effects as a variable to test rather than an established CYP51A1 property. The selected A04032-2 image documents formalin-fixed, paraffin-embedded human prostate carcinoma stained with DAB, but gives no fixation duration or comparison between fixatives (A04032-2 tissue-IHC caption). Compare sections with documented processing histories using the same retrieval, antibody dilution and detection conditions, and score preserved tissue away from damaged edges (standard IHC practice). If staining differs, assess section morphology and processing records before assigning the difference to antigen abundance (standard IHC practice).
Where should convincing CYP51A1 signal appear in chromogenic IHC?
Expect cytoplasmic staining consistent with an endoplasmic reticulum membrane protein, while remembering that DAB cannot resolve the membrane itself (UniProt Q16850 subcellular location; HPA tissue IHC: cytoplasmic Leydig-cell expression; standard IHC practice). CYP51A1 has one annotated transmembrane segment at residues 30–50, which supports checking a cytoplasmic distribution rather than scoring nuclear staining as expected (UniProt Q16850 topology). Compare the pattern with cell morphology and a positive tissue control on the same staining run (standard IHC practice; HPA tissue IHC: High in testis Leydig cells). If signal is chiefly nuclear, extracellular or confined to section edges, review controls, retrieval and detection before interpreting it as CYP51A1 (standard IHC practice).
Can this IHC antibody distinguish CYP51A1 isoforms or reveal a hidden epitope?
The record lists two CYP51A1 isoforms, but the supplied product caption does not map this antibody’s epitope or establish isoform selectivity (UniProt Q16850 isoforms; A04032-2 tissue-IHC caption). Do not assign a positive DAB signal to isoform 1 or 2 without antibody epitope information and an isoform-specific validation experiment (standard IHC interpretation). The protein has one annotated transmembrane segment at residues 30–50; that topology alone cannot identify which side contains the recognized epitope (UniProt Q16850 topology). For weak staining, compare the page’s citrate pH 6.0 retrieval with a carefully controlled time adjustment before inferring that the epitope is absent (page retrieval rule; standard IHC practice).
How can I check CYP51A1 localisation with complementary IF?
Use IF as a complementary localisation check, while keeping chromogenic IHC scoring tied to the paraffin-section workflow (standard IHC/IF practice). In testis, multiplex CYP51A1 with a separately validated Leydig-cell marker and a nuclear counterstain, then compare marker-positive and marker-negative cells (HPA tissue IHC: High in Leydig cells; standard IF practice). Choose a fluorophore channel after inspecting unstained tissue autofluorescence, and include single-stain controls when separating signals (standard IF practice). For permeabilisation, first establish where the antibody epitope lies relative to the ER membrane; the 30–50 transmembrane segment does not establish epitope sidedness, so titrate permeabilisation against morphology and signal (UniProt Q16850 topology; standard IF practice).
What should I adjust when CYP51A1 DAB staining is widespread?
Start with a no-primary control to separate detection background from antibody-dependent staining, and inspect unstained morphology for pigment or deposits (standard IHC practice). For peroxidase-based DAB detection, apply an endogenous peroxidase block and confirm that the no-primary section stays clear under the same development time (A04032-2 tissue-IHC caption: peroxidase/DAB; standard IHC practice). If diffuse signal persists only with primary antibody, titrate its concentration and strengthen the appropriate protein block while keeping retrieval fixed (standard IHC practice). Interpret reduced background against a positive control, because CYP51A1 is reported in the ER and HPA describes cytoplasmic Leydig-cell staining (UniProt Q16850 subcellular location; HPA tissue IHC: Leydig cells).
How should I score CYP51A1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then report the percentage of cytoplasm-positive cells and an H-score based on staining intensity and positive-cell fraction (standard IHC scoring practice; HPA tissue IHC: cytoplasmic Leydig-cell expression). Record the scoring thresholds and keep DAB development, imaging and section selection consistent across samples (standard IHC practice). Normalise counts to the number of eligible cells in each region, or report positive-cell density per mm² when comparing regions with different cellularity (standard IHC quantification practice). Analyse edge damage and necrosis separately, and retain a run-level positive control for detecting technical drift (standard IHC practice).
How can I distinguish genuine CYP51A1 staining from an artefact?
A plausible positive shows cytoplasmic staining in intact cells, consistent with the ER location and the reported Leydig-cell pattern in testis (UniProt Q16850 subcellular location; HPA tissue IHC: cytoplasmic Leydig cells). Review predominantly nuclear or extracellular signal, staining restricted to tissue edges, and deposits over necrosis as possible artefacts before assigning biological meaning (standard IHC interpretation). Compare a no-primary control and an endogenous peroxidase-blocked section to assess detection artefact in the DAB workflow (A04032-2 tissue-IHC caption: peroxidase/DAB; standard IHC practice). HPA flags low consistency with RNA and possible cross-gene antibody targeting, so confirm an unexpected cell pattern with an independent antibody or orthogonal evidence (HPA tissue IHC: reliability description).
Boster reagents
Best CYP51A1 / Lanosterol 14-alpha demethylase IHC Antibodies
The IHC-validated antibody has real data from human formalin-fixed, paraffin-embedded tissue (A04032-2 image caption); the IF/ICC antibody has real data from human CACO-2 cells (A04032-1 image caption).
Real IHC data
Anti-CYP51A1 Antibody (Center)
Cat # A04032-2
Real IF data
Anti-CYP51A1/CYP51 Antibody ®
Cat # A04032-1
A04032-2 supports IHC-P in human tissue, with a DAB image from formalin-fixed, paraffin-embedded prostate carcinoma (catalog applications; A04032-2 image caption). A04032-1 supports IF/ICC, with a fluorescence image from CACO-2 cells (catalog applications; A04032-1 image caption).
Which to pick: Choose A04032-2 for human tissue IHC-P; its own image documents formalin fixation, paraffin embedding and DAB detection, and its listed IHC-P dilution is 1:10–1:50 (A04032-2 image caption; datasheet: 1:10–1:50). Choose A04032-1 for IF/ICC; its CACO-2 image uses 5 μg/mL, while its application list does not include IHC-P (A04032-1 image caption; catalog applications). For work involving mouse or monkey, A04032-1 lists those species as reactive, but its supplied IF image demonstrates human cells only (A04032-1 catalog reactivity; A04032-1 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16850 (CP51A_HUMAN, Lanosterol 14-alpha demethylase).
- Human Protein Atlas. CYP51A1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CYP51A1 subcellular location (ICC-IF): Localized to the endoplasmic reticulum. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. CYP51A1 antibody validation summary (2 antibodies).
- Machine Learning-based Framework Develops a Tumor Thrombus Coagulation Signature in Multicenter Cohorts for Renal Cancer. International journal of biological sciences 2024 — PMC11234220.
- Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer. Oncotarget 2016 — PMC5216813.
- Atorvastatin inhibits pancreatic carcinogenesis and increases survival in LSL-KrasG12D-LSL-Trp53R172H-Pdx1-Cre mice. Molecular carcinogenesis 2013 — PMC3411900.
- PubMed PMID:8619637 — UniProt-cited evidence.
- PubMed PMID:8797093 — UniProt-cited evidence.
- PubMed PMID:8809088 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYP51A1 (Lanosterol 14-alpha demethylase). Expected localisation Leydig-cell cytoplasm (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: A04032-2 at 1:10-1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: FFPE human prostate carcinoma tissue (datasheet A04032-2); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A04032-2); 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-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-CYP51A1, 1:10-1:50 (datasheet A04032-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Use formalin-fixed paraffin sections (datasheet: A04032-2 IHC-P) (selected-SKU IHC image A04032-2); keep fixation consistent. (standard IHC practice; not target-specific); Caveat: Antibody may detect protein from another gene (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unreported (UniProt); Regulation: Expression varies by tissue (UniProt). Evidence: UniProt Q16850, HPA tissue IHC · Approved, HPA ICC-IF · endoplasmic reticulum. Caution: Based on antibodies targeting…, Datasheet A04032-2, PMC5216813. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYP51A1/immunohistochemistry-ihc-guide.