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CYP26B1 / Cytochrome P450 26B1 · IHC design guide
Design Immunohistochemistry for CYP26B1
Plan paraffin-section CYP26B1 IHC using the observed cytoplasmic tissue pattern and the protein’s ER location (HPA tissue IHC; UniProt). Compare staining with tissue controls and interpret intensity cautiously because tissue-IHC reliability is uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYP26B1 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 staining (HPA tissue IHC); ER and microsomal membrane location (UniProt) | |
| Staining pattern | Cytoplasmic staining in several tissues, including Purkinje cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02691-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | Staining has low agreement with RNA data; reliability is uncertain (HPA tissue IHC) | |
| Regulation | RNA is tissue enhanced in brain and skin (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; effects on epitope recognition are unknown (UniProt) |
Section 1
Recommended CYP26B1 IHC & IF Protocols
The catalog antibody protocol (datasheet A02691-2) is accompanied by a published oral mucosa IHC protocol (PMC13304650).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A02691-2) |
| Fixation | Image fixative and duration unreported (datasheet A02691-2); 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 A02691-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02691-2) |
| Primary antibody | Rabbit anti-CYP26B1, 2-5 μg/ml (datasheet A02691-2) |
| Primary incubation | Overnight at 4 °C (datasheet A02691-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02691-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CYP26B1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A02691-2); the published IHC method specifies heat-induced retrieval without a buffer (PMC13304650).
Section 2
What Is the Expected CYP26B1 Staining Pattern?
CYP26B1 is annotated at the endoplasmic reticulum and microsome membranes, with no transmembrane segment annotated (UniProt Q9NR63: location and topology). In paraffin sections, expect cytoplasmic staining in selected cells, including cerebellar Purkinje cells and liver hepatocytes (HPA tissue IHC: Medium). Treat this pattern as provisional: HPA rates tissue IHC reliability Uncertain because antibody staining has low consistency with RNA expression (HPA: reliability).
| Selective cytoplasmic staining in Purkinje cells, hepatocytes, or adrenal glandular cells, with identifiable cell boundaries. | This fits the reported compartment and cell types; HPA scores each example Medium, so a strong signal is not required for a plausible positive. Morphology and negative controls still matter because the tissue IHC assessment is Uncertain (HPA tissue IHC: profile, levels, reliability). |
| Signal is confined to nuclei, extracellular material, or another compartment without discernible cytoplasmic staining. | That distribution does not match HPA tissue IHC's cytoplasmic profile or UniProt's endoplasmic reticulum and microsome membrane annotations (HPA tissue IHC: profile; UniProt Q9NR63: location). Review counterstain, focus, and detection controls before assigning it to CYP26B1 (general IHC practice). |
| Prominent staining appears in adipocytes or cardiomyocytes, while the expected positive cells show little signal. | HPA reports CYP26B1 as Not detected in those cell types (HPA tissue IHC: adipose tissue, heart muscle). Cross-reactivity or endogenous detection activity is possible; compare a no-primary control and a known-positive section processed together (general IHC practice). |
| Uniform color covers many cell types, vessels, and extracellular spaces, obscuring cell outlines. | This is difficult to score as selective cytoplasmic staining (HPA tissue IHC: cytoplasmic profile). Nonspecific binding, incomplete blocking, or detection background can produce this appearance; examine the no-primary control and localize the precipitate before interpreting the slide (general IHC practice). |
| No convincing stain is visible in cerebellar Purkinje cells or liver hepatocytes. | Both are reported at Medium staining, making them useful comparison cells (HPA tissue IHC: cerebellum, liver). A blank result can reflect staining workflow failure or sample variation; it does not alone overturn the HPA pattern, whose reliability is Uncertain (HPA: reliability; general IHC practice). |
💡Expected CYP26B1 appearanceCall a result positive when selected cells show discernible cytoplasmic, typically Medium staining—such as Purkinje cells or hepatocytes—while nuclear-only or diffuse tissue-wide color warrants control review (HPA tissue IHC: profile, levels; general IHC practice).
| Compartment evidence | UniProt places CYP26B1 at endoplasmic reticulum and microsome membranes; HPA tissue IHC describes cytoplasmic staining (UniProt Q9NR63: location; HPA tissue IHC: profile). Score the visible cytoplasmic pattern rather than requiring a resolvable membrane outline in chromogenic sections (general IHC practice). |
| Tissue evidence and confidence | HPA reports Medium staining in several distinct cell types, but rates overall tissue IHC Uncertain because staining and RNA data have low consistency (HPA tissue IHC: positive cells, reliability). Use cell-specific comparisons and controls when interpreting an unexpected positive. |
| Antibody validation | The listed rabbit polyclonal HPA012567 is rated Uncertain for IHC and Approved for ICC (HPA antibodies: HPA012567). The ICC rating does not establish reliable paraffin-section staining; interpret the IHC result with the tissue evidence and controls. |
| Molecular annotations | UniProt lists 3 isoforms, no annotated transmembrane segment, no signal peptide or propeptide, and no glycosylation sites (UniProt Q9NR63: isoforms, topology, processing, glycosylation). These annotations do not identify the antibody epitope or establish whether it detects every isoform. |
| IF/ICC Q: What location is reported? | A: HPA reports approved cytosol localisation and lists ICC-IF images for A-431, U-251MG, and U2OS (HPA subcellular: location, image list). This observation comes from ICC-IF; it does not resolve the uncertainty of tissue IHC (HPA tissue IHC: reliability). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive tissue is blank. | The stain may have failed, or the sampled cells may differ from the reported pattern; HPA rates Purkinje cells and hepatocytes Medium (HPA tissue IHC: cerebellum, liver). | Confirm the expected cells are present, then check the positive control and review retrieval, antibody incubation, and detection steps (general IHC practice). |
| Color is diffuse across the section. | Nonspecific binding or detection background may obscure HPA's reported cytoplasmic, cell-selective pattern (HPA tissue IHC: profile; general IHC practice). | Compare the no-primary control, check blocking and washes, and reduce antibody concentration if the assay controls support that adjustment (general IHC practice). |
| Only nuclei are stained. | Nuclear-only signal is discordant with the reported cytoplasmic tissue profile and endoplasmic reticulum or microsome location (HPA tissue IHC: profile; UniProt Q9NR63: location). | Verify nuclear identity with the counterstain, inspect controls, and avoid scoring nuclear-only color as a CYP26B1 positive (general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly. | Those cells are reported Not detected; unexpected color may reflect cross-reactivity or detection background (HPA tissue IHC: adipose tissue, heart muscle; general IHC practice). | Run a no-primary control alongside a reported positive tissue and compare staining by cell type before assigning specificity (general IHC practice). |
| Color persists when the primary antibody is omitted. | The signal arises from the detection workflow rather than primary-antibody binding; endogenous enzyme activity is one possibility with enzyme-based detection (general IHC practice). | Check the detection chemistry and its blocking step; repeat the no-primary control under the same development conditions (general IHC practice). |
| An ICC-IF image looks broadly cytosolic, but the paraffin section is ambiguous. | HPA approves the ICC-IF cytosol location while rating tissue IHC Uncertain; the two assessments have different evidence (HPA subcellular: location; HPA tissue IHC: reliability). | Interpret the paraffin section against its own cell-specific tissue pattern and IHC controls; use the separate IF/ICC guide for that application's workflow. |
Experiment evidence
IHC & IF images showing CYP26B1 staining
A02691-2 · IHCIHC analysis of CYP26B1 using anti-CYP26B1 antibody (A02691-2). CYP26B1 was detected in a paraffin-embedded section of human liver cancer ti
A02691-2 · IHCIHC analysis of CYP26B1 using anti-CYP26B1 antibody (A02691-2). CYP26B1 was detected in a paraffin-embedded section of human thyroid cancer
A02691-2 · IFIF analysis of CYP26B1 using anti-CYP26B1 antibody (A02691-2). CYP26B1 was detected in an immunocytochemical section of U2OS cells. Enzyme a
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000003137-CYP26B1/tissue/cerebral+cortex#imid_3391420
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000003137-CYP26B1/tissue/cerebellum#imid_3391423
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000003137-CYP26B1/tissue/colon#imid_3391338
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000003137-CYP26B1/tissue/liver#imid_3391331
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA012567; green = CYP26B1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA012567; green = CYP26B1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYP26B1 IHC & IF
🧪Run cerebellum first and look for CYP26B1 staining in Purkinje cells (HPA: cerebellum, Purkinje cells, Medium); use esophagus as the negative tissue and assess its squamous epithelial cells (HPA: esophagus, squamous epithelial cells, Not detected). On the cerebellum slide, use neighboring non-Purkinje cells as internal background comparators and expect little or no staining relative to Purkinje cells; the supplied HPA row does not establish those neighboring cells as a validated negative (HPA: cerebellum, Purkinje cells, Medium).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYP26B1 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched nonimmune rabbit IgG isotype control, and CYP26B1 knockout material as a biological negative if available (A02691-2 caption: rabbit primary antibody). For cerebellar IHC, quench endogenous peroxidase and inspect background before interpreting HRP/DAB staining (A02691-2 caption: HRP/DAB detection; HPA: cerebellum, Purkinje cells, Medium).
⚠️Feasibility: The selected A02691-2 paraffin-section caption leaves the fixative unreported, and the supplied evidence reports no target-specific fixation window or fixation effect (A02691-2 caption). Heat-mediated retrieval in EDTA at pH 8.0 was used in that caption; whether retrieval is essential remains unreported (A02691-2 caption). The supplied evidence does not establish that frozen sections or IF/ICC are easier; if using IF in cerebellum, check autofluorescence and assess localisation cautiously because HPA reports cytosol while UniProt reports ER and microsome membrane (HPA subcellular; UniProt Q9NR63 subcellular location).
HPA tissue IHC evidence for CYP26B1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CYP26B1 IHC Tips
Troubleshoot CYP26B1 chromogenic IHC in paraffin sections using the catalog antibody’s tissue image, protein annotation, and cautiously interpreted reference staining.
What retrieval should I start with if CYP26B1 staining is weak?
Start with heat-mediated EDTA retrieval at pH 8.0 (datasheet A02691-2). The catalog antibody’s paraffin-section image used that condition, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A02691-2). If signal remains weak, compare a modestly longer retrieval against the starting condition on adjacent sections while holding antibody concentration and DAB development constant (standard IHC practice). Check that any gain occurs in plausible cytoplasmic staining rather than section edges or damaged areas (HPA: cytoplasmic tissue expression; standard IHC practice). Record retrieval time and section integrity for each condition so stronger staining is not mistaken for better specificity (standard IHC practice).
Could fixation explain weak CYP26B1 staining in my paraffin sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report its fixative (datasheet A02691-2). Do not treat its EDTA pH 8.0 retrieval result as evidence that a particular fixation duration preserves CYP26B1 (datasheet A02691-2). For troubleshooting, compare sections with documented processing histories, checking morphology, tissue retention, and staining in the same run (standard IHC practice). Keep retrieval, 2 μg/ml primary concentration, and DAB development comparable when assessing a processing effect (datasheet A02691-2; standard IHC practice). If fixation history is unavailable, report that limitation alongside the staining result rather than assigning a target-specific cause (standard IHC practice).
Should CYP26B1 appear cytoplasmic or confined to an ER pattern in IHC?
Expect staining within the cytoplasmic compartment, while allowing for an ER-associated distribution: CYP26B1 is annotated at the endoplasmic reticulum and microsome membranes, and HPA describes cytoplasmic tissue staining (UniProt Q9NR63; HPA: tissue IHC profile). HPA separately reports approved cytosolic localisation from ICC/IF, so these sources do not establish a single resolvable IHC pattern (HPA: subcellular). The protein has no annotated transmembrane segment, which does not by itself identify the antibody epitope’s membrane-facing side (UniProt Q9NR63 topology). Evaluate distribution in intact cells against the no-primary control and morphology (standard IHC practice). Treat predominantly nuclear or extracellular DAB as a specificity concern requiring independent checks (UniProt Q9NR63 localisation; standard IHC practice).
Can this IHC antibody distinguish CYP26B1 isoforms or require a particular permeabilisation route?
CYP26B1 has 3 annotated isoforms, but the supplied product evidence does not map this antibody’s epitope to any of them (UniProt Q9NR63 isoforms; datasheet A02691-2). Its absence of an annotated transmembrane segment and of annotated glycosylation sites does not establish epitope accessibility after paraffin processing (UniProt Q9NR63 topology and glycosylation). Use the documented EDTA pH 8.0 retrieval as the initial IHC condition, then assess staining with an appropriate negative control (datasheet A02691-2; standard IHC practice). If isoform attribution matters, obtain epitope or sequence-specific validation before assigning a stained cell to isoform 1, 2, or 3 (UniProt Q9NR63 isoforms; standard IHC practice).
How can IF help check an ambiguous CYP26B1 IHC pattern?
Use IF as a separate cross-check of cell identity and compartment when chromogenic IHC is ambiguous (standard IHC/IF practice). Multiplex CYP26B1 with a validated marker for the expected cell type, such as Purkinje cells in cerebellum, and examine overlap within individual cells (HPA: medium staining in cerebellar Purkinje cells; standard IF practice). Choose spectrally separated fluorophores and consider a far-red channel after checking tissue autofluorescence and single-stain controls (standard IF practice). Because the epitope’s membrane-facing side is unspecified, establish permeabilisation empirically without assuming that the ER annotation identifies it (UniProt Q9NR63 localisation and topology; standard IF practice). Compare the result with IHC cautiously: HPA reports cytosolic ICC/IF localisation, while its tissue-IHC reliability is uncertain (HPA: subcellular; HPA: tissue IHC reliability).
How do I reduce diffuse brown background without losing CYP26B1 signal?
First compare a no-primary control with the stained section to separate detection-system background from antibody-dependent colour (standard IHC practice). The selected image used a 10% goat-serum block, 2 μg/ml rabbit primary overnight at 4°C, and DAB detection (datasheet A02691-2). Check endogenous peroxidase blocking before the HRP step, then compare shorter DAB development and a lower primary concentration on matched sections (standard chromogenic IHC practice). Keep EDTA retrieval at pH 8.0 initially so the background comparison remains interpretable (datasheet A02691-2; standard IHC practice). Reject diffuse edge or necrotic-area colour as evidence of CYP26B1 without intact-cell localisation and clean controls (UniProt Q9NR63 localisation; standard IHC practice).
How should I score CYP26B1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score the fraction of cytoplasm-positive cells and staining intensity within a defined cell population; an H-score combines those observations if the same scoring rules are used throughout (HPA: cytoplasmic tissue expression; standard IHC practice). Alternatively, report percent-positive cells or positive-cell density per mm² of viable tissue, with the denominator specified (standard IHC practice). Normalize comparisons to the same cell type, sampled area, retrieval condition, and DAB development time (standard IHC practice). Exclude necrosis, folds, and section edges before calculating scores (standard IHC practice). Because HPA rates its tissue-IHC pattern uncertain, report control performance and observer or threshold rules alongside any between-sample difference (HPA: tissue IHC reliability; standard IHC practice).
When is CYP26B1 staining credible rather than an artefact?
A credible result shows staining in intact cells within a plausible cytoplasmic compartment and exceeds the matched no-primary control (UniProt Q9NR63 localisation; HPA: cytoplasmic tissue expression; standard IHC practice). Cerebellar Purkinje cells and hepatocytes are examples with medium HPA staining, but that atlas pattern has uncertain reliability and cannot validate a new specimen by itself (HPA: tissue IHC). Scrutinize predominantly nuclear or extracellular colour, section-edge gradients, necrotic regions, and signal present without primary antibody (UniProt Q9NR63 localisation; standard IHC practice). Check endogenous peroxidase and DAB development before calling diffuse brown colour positive (standard chromogenic IHC practice). Interpret differences in staining as protein-distribution observations, not direct measurements of retinoic-acid metabolism (UniProt Q9NR63 function; standard IHC practice).
Boster reagents
Best CYP26B1 / Cytochrome P450 26B1 IHC Antibodies
A02691-2 has IHC images from paraffin-embedded human liver and thyroid cancer tissue and an IF/ICC image from U2OS cells (catalog image captions).
Real IHC data
Anti-CYP26B1 Antibody ®
Cat # A02691-2
A02691-2 is listed for IHC and IF/ICC, with human, mouse and rat reactivity (catalog applications and reactivity). Its IHC images show human liver and thyroid cancer tissue; its IF/ICC image shows U2OS cells (catalog image captions).
Which to pick: For tissue IHC, choose A02691-2: its captions show EDTA pH 8 retrieval and DAB detection in paraffin sections of human liver and thyroid cancer tissue; the fixative is unreported (catalog IHC captions). For IF/ICC, the same SKU has an image from U2OS cells (catalog IF/ICC caption). For mouse or rat samples, A02691-2 lists both species as reactive, but the supplied IHC and IF/ICC images do not demonstrate either species; clonality is unreported (catalog reactivity and image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NR63 (CP26B_HUMAN, Cytochrome P450 26B1).
- Human Protein Atlas. CYP26B1 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CYP26B1 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. CYP26B1 antibody validation summary (1 antibodies).
- Prognostic implication and immunotherapy response prediction of a novel ubiquitination-related gene signature in liver cancer. Aging 2024 — PMC11210240.
- Retinoic acid metabolism related gene CYP26B1 promotes tumor stemness and tumor microenvironment remodeling in bladder cancer. Journal of Cancer 2025 — PMC12170498.
- 5-Methoxyleoligin, a lignan from Edelweiss, stimulates CYP26B1-dependent angiogenesis in vitro and induces arteriogenesis in infarcted rat hearts in vivo. PloS one 2013 — PMC3595277.
- Connective tissue profiling in keratinized and non-keratinized oral mucosa reveals distinct extracellular and intracellular features. Frontiers in cell and developmental biology 2026 — PMC13304650.
- PubMed PMID:10823918 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYP26B1 (Cytochrome P450 26B1). Expected localisation Cytoplasmic staining (HPA tissue IHC); ER and microsomal membrane location (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02691-2). Validated IHC antibody: A02691-2 at 2-5 μg/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 cancer tissue; fixative not specified (datasheet A02691-2); 2. Fixation: Image fixative and duration unreported (datasheet A02691-2); 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 A02691-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02691-2); 8. Primary antibody: Rabbit anti-CYP26B1, 2-5 μg/ml (datasheet A02691-2); 9. Primary incubation: Overnight at 4 °C (datasheet A02691-2). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Caveat: Staining has low agreement with RNA data; reliability is uncertain (HPA tissue IHC); Isoform / epitope: 3 isoforms; effects on epitope recognition are unknown (UniProt); Regulation: RNA is tissue enhanced in brain and skin (HPA tissue RNA). Evidence: UniProt Q9NR63, HPA tissue IHC · Uncertain, HPA ICC-IF · cytosol., Datasheet A02691-2, PMC13304650. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYP26B1/immunohistochemistry-ihc-guide.