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CYP17A1 / Steroid 17-alpha-hydroxylase/17,20 lyase · IHC design guide
Design Immunohistochemistry for CYP17A1
Plan CYP17A1 IHC in paraffin sections using adrenal zona fasciculata cells and testicular Leydig cells as positive references (HPA tissue IHC). The catalog antibody was used at 1 μg/ml on human testis sections (datasheet A00615-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYP17A1 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 | ER and microsomal membrane; tissue-IHC compartment unconfirmed (UniProt) | |
| Staining pattern | High staining in adrenal zona fasciculata and Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A00615-1) | |
| Positive control | Adrenal gland+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep tissue fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms or processing; epitope unknown (UniProt) |
Section 1
Recommended CYP17A1 IHC & IF Protocols
The catalog antibody’s citrate pH 6 IHC-P protocol (datasheet A00615-1) is accompanied by four published adrenal IHC protocols (PMC10512174; PMC12185499; PMC5638684; PMC7460193).
| Sample | Paraffin-embedded human testis tissues; fixative not specified (datasheet A00615-1) |
| Fixation | Image fixative and duration unreported (datasheet A00615-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A00615-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00615-1) |
| Primary antibody | Rabbit anti-CYP17A1, 0.5-1μg/ml (datasheet A00615-1) |
| Primary incubation | Overnight at 4 °C (datasheet A00615-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00615-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CYP17A1-positive staining in cells in zona fasciculata of adrenal gland (HPA tissue IHC: High). HPA tissue profile: High expression in Leydig cells and adrenal cotical cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A00615-1). The published EDTA pH 9 condition used a different antibody (PMC7460193 methods).
Section 2
What Is the Expected CYP17A1 Staining Pattern?
CYP17A1 is assigned to the endoplasmic reticulum and microsome membranes, with no transmembrane segment annotated (UniProt P05093: location and topology). In IHC, expect staining in adrenal zona fasciculata cells and testicular Leydig cells (HPA: High in both). HPA rates the tissue staining Enhanced, while describing only medium consistency between antibody staining and RNA expression (HPA: reliability).
| Adrenal zona fasciculata cells or testicular Leydig cells show strong, predominantly cytoplasmic staining. | This fits the reported High staining in those cells (HPA: tissue IHC). A cytoplasmic, ER-associated distribution is consistent with the protein location (UniProt P05093: ER/microsome membrane). Assess the named cell population, rather than treating all cells in the section as equally positive. |
| The dominant signal is confined to nuclei in an otherwise plausible positive tissue. | A nuclear-only pattern conflicts with the annotated ER and microsome membrane location (UniProt P05093: subcellular location). Check the counterstain and a no-primary control before interpreting it as CYP17A1; nuclear chromogen can reflect nonspecific staining or a reading error (general IHC practice). |
| Strong staining appears chiefly in an unexpected cell population, such as adrenal cells outside the identified positive population. | The supplied positive call is for zona fasciculata cells, and the testis call is for Leydig cells (HPA: tissue IHC). Staining elsewhere does not establish CYP17A1 expression by itself; consider antibody cross-reactivity or endogenous detection activity and compare controls (general IHC practice). |
| Color is spread across many cell types or extracellular areas without a clear cellular pattern. | Diffuse background cannot be scored as the reported cell-specific pattern (HPA: High in zona fasciculata and Leydig cells). Uneven blocking, residual detection reagent, or endogenous activity are possible general IHC causes; examine a no-primary control and the distribution of the chromogen (general IHC practice). |
| Neither zona fasciculata cells nor Leydig cells stain in an otherwise interpretable section. | An absent signal conflicts with the reported High IHC staining in those named cells (HPA: tissue IHC), but a single negative slide cannot distinguish technical failure from sample variation. Confirm the cell identity and run a known-positive section alongside the test section (general IHC practice). |
💡Expected CYP17A1 appearanceCall a section positive when the named adrenal zona fasciculata or testicular Leydig cells show conspicuous cytoplasmic staining consistent with ER-associated CYP17A1 (HPA: High in both; UniProt P05093: ER/microsome membrane); isolated nuclear or widespread cell-independent color warrants control review (general IHC practice).
| Cell and tissue context | The strongest supplied IHC anchors are zona fasciculata cells and Leydig cells (HPA: High). Adipocytes in adipose tissue and appendix glandular cells are reported Not detected (HPA: tissue IHC); these are context checks, not universal negative controls for every assay. |
| Compartment assignment | ER and microsome membrane localisation supports an intracellular, predominantly cytoplasmic interpretation (UniProt P05093: location). The record annotates no transmembrane segment (UniProt P05093: topology), so do not infer a cell-surface staining pattern from the word membrane. |
| Strength of tissue evidence | The antibody has Enhanced IHC validation, but HPA also reports medium agreement between staining and RNA expression (HPA: antibody validation and reliability). Use the reported cell pattern as a benchmark, while resolving unexpected results with matched controls. |
| IF/ICC Q: Should vesicles define the IHC-P result? | A: HPA reports an approved vesicular ICC-IF location (HPA: subcellular ICC-IF), whereas UniProt assigns ER and microsome membranes (UniProt P05093: location). The IF observation belongs to a different assay and does not establish an IHC-P staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in a known-positive adrenal or testis section. | The expected positive cells may be absent from the sampled area, or an IHC step may have failed (HPA: High in zona fasciculata and Leydig cells; general IHC practice). | Verify the named cells on the section; repeat with a concurrent known-positive section and check the catalog antibody's IHC-P instructions, detection reagents, and counterstain (general IHC practice). |
| Only nuclei show strong chromogen. | This conflicts with ER/microsome localisation (UniProt P05093: location); nonspecific signal or counterstain confusion is possible (general IHC practice). | Compare a no-primary control and review chromogen separately from the counterstain; score CYP17A1 only after the cellular compartment is clear (general IHC practice). |
| Many unrelated cells stain as strongly as the expected positive cells. | The pattern exceeds the named High populations (HPA: tissue IHC); antibody cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Compare no-primary and known-positive controls, then reassess cell identity and detection background before assigning expression (general IHC practice). |
| Background obscures the borders of positive cells. | General IHC causes include incomplete blocking, residual reagent, or excessive chromogen development (general IHC practice). | Inspect the no-primary control and optimize blocking, washing, or development against a concurrent positive section; do not score diffuse color as cellular staining (general IHC practice). |
| An HPA Not detected cell type appears positive. | Adipocytes in adipose tissue and appendix glandular cells are reported Not detected (HPA: tissue IHC); the discrepancy may reflect technical background or a different sample context. | Check morphology and controls, then describe the observed result without treating a single HPA negative call as proof that all samples must be negative (general IHC interpretation). |
| IHC looks cytoplasmic, while an ICC-IF image looks vesicular. | The sources report different location descriptions in different assays (UniProt P05093: ER/microsome membrane; HPA: approved vesicular ICC-IF). | Interpret the paraffin-section IHC against its named positive cells and controls (HPA: tissue IHC; general IHC practice); assess ICC-IF on its separate guide page. |
Experiment evidence
IHC & IF images showing CYP17A1 staining
A00615-1 · IHCIHC analysis of CYP17A1 using anti-CYP17A1 antibody (A00615-1). CYP17A1 was detected in paraffin-embedded section of human testis tissues. H
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000148795-CYP17A1/tissue/cerebral+cortex#imid_17610351
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000148795-CYP17A1/tissue/adrenal+gland#imid_417
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000148795-CYP17A1/tissue/colon#imid_17610291
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000148795-CYP17A1/tissue/liver#imid_17610284
HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA048533; green = CYP17A1, blue = nucleus, red = microtubules.
HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA048533; green = CYP17A1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYP17A1 IHC & IF
🧪Run testis first: Leydig cells should stain strongly (HPA: High in testis Leydig cells; selected A00615-1 caption: testis IHC). Use adipose tissue as the negative, with adipocytes expected to lack staining (HPA: Not detected in adipocytes); non-Leydig cells on the testis slide should provide an internal comparison for background staining (HPA: High staining is assigned to Leydig cells).
Positive control tissue: Adrenal gland (Cells in zona fasciculata, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYP17A1 in OE19, SH-SY5Y, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality where known (selected A00615-1 caption: rabbit primary; standard IHC practice). A validated CYP17A1 knockout specimen or antigen peptide competition can test specificity; for the caption’s biotin-based DAB method, block endogenous peroxidase and assess endogenous biotin staining in testis (selected A00615-1 caption: SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A00615-1 paraffin-section caption does not state the fixative (selected A00615-1 caption: fixative unreported). The reported testis method uses citrate retrieval at pH 6 for 20 minutes, but whether CYP17A1 staining depends on that retrieval is unreported (selected A00615-1 caption: citrate retrieval). The supplied evidence does not establish that frozen sections or IF are easier; endogenous peroxidase or biotin may complicate interpretation of the biotin-based DAB stain in testis (selected A00615-1 caption: SABC and DAB; standard IHC practice).
HPA tissue IHC evidence for CYP17A1
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 | Cells in zona fasciculata | High | Protein (IHC) | HPA → |
| 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 CYP17A1 IHC Tips
Use cell type and subcellular pattern alongside matched controls when interpreting CYP17A1 staining (HPA tissue IHC; UniProt P05093).
How should I optimize retrieval when CYP17A1 staining is weak in paraffin sections?
Start with heat mediated retrieval in citrate buffer at pH 6 for 20 minutes, as used for A00615-1 (datasheet A00615-1; catalog IHC caption). Compare freshly cut positive control sections of adrenal gland or testis across runs, keeping heating, cooling and detection conditions consistent (HPA tissue IHC; standard IHC practice). If staining remains weak, adjust heating duration in small increments and check tissue preservation before trying another retrieval buffer as a fallback (standard IHC practice). Score signal in adrenal zona fasciculata cells or testicular Leydig cells, where expression is high, rather than judging intensity across the whole section (HPA tissue IHC).
Could fixation explain weak or uneven CYP17A1 staining?
Target specific fixation sensitivity is unknown because the A00615-1 tissue caption identifies paraffin embedded human testis but does not report its fixative (catalog IHC caption: A00615-1). Record the actual fixative, fixation interval and tissue thickness for each specimen, then compare sections processed under matched conditions (standard IHC practice). Overfixation, underfixation and uneven penetration can affect morphology or antigen accessibility in IHC generally; none is established here as a CYP17A1 specific effect (standard IHC practice). If signal varies between blocks, repeat the citrate pH 6 retrieval with a known positive control in the same run before attributing the difference to fixation (datasheet A00615-1; HPA tissue IHC; standard IHC practice).
What cellular staining pattern should count as plausible CYP17A1 signal?
Expect predominantly cytoplasmic staining compatible with endoplasmic reticulum and microsome membranes, since CYP17A1 is assigned to those locations (UniProt P05093). The record lists no transmembrane segment, so a sharp cell surface outline alone would need independent validation rather than being assumed to represent CYP17A1 (UniProt P05093; standard IHC practice). In tissue, prioritize Leydig cells and adrenal zona fasciculata cells, both reported with high staining (HPA tissue IHC). A vesicular IF pattern has also been reported, but compartment calls from cultured cell imaging should be checked against tissue morphology before interpreting puncta in chromogenic sections (HPA subcellular; standard IHC practice).
How do unknown epitope details affect retrieval and antibody interpretation?
The supplied record annotates 0 isoforms and no glycosylation sites or modified residues, but it does not map the A00615-1 epitope (UniProt P05093; catalog IHC caption: A00615-1). Do not assign a membrane facing side or infer that a negative paraffin section reflects epitope masking from these annotations alone (UniProt P05093; standard IHC practice). Keep the catalog antibody's citrate pH 6 retrieval as the initial condition, then compare retrieval changes against a positive control and a no primary control (datasheet A00615-1; standard IHC practice). If another antibody gives a different pattern, check its documented immunogen and tissue validation before claiming an isoform specific result (standard IHC practice).
How should I adapt CYP17A1 staining for multiplex immunofluorescence?
Treat IF as a separate optimization because the supplied A00615-1 evidence describes chromogenic paraffin section IHC, not an IF validation (catalog IHC caption: A00615-1). Pair CYP17A1 with a validated marker of Leydig cells or adrenal zona fasciculata cells, then confirm that any apparent overlap remains within the expected cell population (HPA tissue IHC; standard IF practice). Select fluorophores after measuring tissue autofluorescence and reserve a spectrally distinct, brighter channel for weak CYP17A1 signal (standard IF practice). Because the antibody epitope's membrane facing side is unspecified, titrate permeabilisation and verify access with appropriate controls instead of assuming that the UniProt membrane annotation identifies the accessible side (UniProt P05093; standard IF practice).
What should I change if DAB background obscures CYP17A1 positive cells?
First inspect no primary and secondary only controls to distinguish detection background from antibody dependent staining (standard IHC practice). For a peroxidase DAB workflow, block endogenous peroxidase and optimize washing and chromogen development; these are general IHC steps, not CYP17A1 specific evidence (standard IHC practice). The A00615-1 caption used 10% goat serum blocking, 1 µg/mL primary antibody overnight at 4 °C, and biotinylated secondary followed by complex based DAB detection (catalog IHC caption: A00615-1). If background persists, titrate primary concentration and evaluate endogenous biotin or other detection system background while retaining Leydig cell staining in the positive control (HPA tissue IHC; standard IHC practice).
How can I compare CYP17A1 staining between paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, using Leydig cells in testis or zona fasciculata cells in adrenal gland when those tissues are studied (HPA tissue IHC; standard IHC practice). Report the percentage of positive target cells and an H score from 0–300 using intensity grades 0–3, or report positive cell density per mm² when cell counts are the endpoint (standard IHC practice). Normalize cell based measures to the number of morphologically eligible cells and area based measures to analyzed viable tissue area (standard IHC practice). Apply the same threshold, counterstain criteria, retrieval and imaging settings across samples, with a positive control in each run (standard IHC practice).
How can I distinguish true CYP17A1 staining from tissue artefacts?
A convincing positive signal should occupy the expected steroidogenic cells and show a cytoplasmic pattern compatible with endoplasmic reticulum or microsome localization (HPA tissue IHC; UniProt P05093). High staining is reported in testicular Leydig cells and adrenal zona fasciculata cells, whereas adipocytes in adipose tissue were not detected in the supplied tissue profile (HPA tissue IHC). Flag isolated nuclear or purely surface staining, section edge enhancement and staining in necrotic areas for review against morphology and controls (UniProt P05093; standard IHC practice). Check no primary and peroxidase controls when DAB signal could reflect endogenous enzyme activity, and avoid treating intensity alone as proof of steroidogenic function (standard IHC practice; UniProt P05093).
Boster reagents
Best CYP17A1 / Steroid 17-alpha-hydroxylase/17,20 lyase IHC Antibodies
The IHC-validated antibody has a human testis paraffin-section image (A00615-1 image caption); the catalog also lists IF/ICC use in human, mouse, and rat (M00615-1 applications/reactivity).
Real IHC data
Anti-Cytochrome P450 17A1/CYP17A1 Antibody ®
Cat # A00615-1
A00615-1 is the SKU with a rendered IHC card: its image shows CYP17A1 staining in a human testis paraffin section (A00615-1 image caption). M00615-1 lists IHC and IF/ICC applications and human, mouse, and rat reactivity, but has no IHC or IF image in the payload (M00615-1 applications/reactivity/image alts).
Which to pick: For paraffin-section IHC, choose A00615-1 because its own image documents human testis staining after citrate retrieval; the fixative is unreported (A00615-1 image caption). For IF/ICC or mouse samples, consider rabbit monoclonal M00615-1 based on its listed applications and reactivity, while noting that no IF/ICC or IHC image is supplied for it (M00615-1 host/clone/applications/reactivity/image alts). For human or rat IHC, A00615-1 lists both species as reactive, although its pictured paraffin section is human only (A00615-1 reactivity/image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P05093 (CP17A_HUMAN, Steroid 17-alpha-hydroxylase/17,20 lyase).
- Human Protein Atlas. CYP17A1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CYP17A1 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. CYP17A1 antibody validation summary (1 antibodies).
- Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness. Frontiers in endocrinology 2023 — PMC10512174.
- Histopathological spectrum of common aldosterone-driver gene mutations in aldosterone-producing adenomas. Frontiers in medicine 2025 — PMC12185499.
- Histopathological and genetic characterization of aldosterone-producing adenomas with concurrent subclinical cortisol hypersecretion: a case series. Endocrine 2017 — PMC5638684.
- Incomplete Pattern of Steroidogenic Protein Expression in Functioning Adrenocortical Carcinomas. Biomedicines 2020 — PMC7460193.
- PubMed PMID:3025870 — UniProt-cited evidence.
- PubMed PMID:3500022 — UniProt-cited evidence.
- PubMed PMID:3274893 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYP17A1 (Steroid 17-alpha-hydroxylase/17,20 lyase). Expected localisation ER and microsomal membrane; tissue-IHC compartment unconfirmed (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A00615-1). Validated IHC antibody: A00615-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Cells in zona fasciculata); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human testis tissues; fixative not specified (datasheet A00615-1); 2. Fixation: Image fixative and duration unreported (datasheet A00615-1); 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: Citrate pH 6 , 20 min (datasheet A00615-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00615-1); 8. Primary antibody: Rabbit anti-CYP17A1, 0.5-1μg/ml (datasheet A00615-1); 9. Primary incubation: Overnight at 4 °C (datasheet A00615-1). Watch for: Fixation: Keep tissue fixation consistent. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms or processing; epitope unknown (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt P05093, HPA tissue IHC · Enhanced, HPA ICC-IF · vesicles., Datasheet A00615-1, PMC10512174, PMC12185499. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYP17A1/immunohistochemistry-ihc-guide.