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BTD / Biotinidase · IHC design guide
Design Immunohistochemistry for BTD
Plan chromogenic BTD IHC on paraffin sections using catalog antibody A03020 at 1:100–1:200 (datasheet: 1:100–1:200). Assess endothelial membranous staining alongside high staining in breast myoepithelial cells and kidney glomerular cells, while accounting for uncertain agreement between staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BTD 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 | Endothelial membranes (HPA tissue IHC); extracellular space (UniProt) | |
| Staining pattern | Membranous staining in endothelial cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+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 fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted BTD may separate RNA and staining sites (HPA tissue IHC) | |
| Regulation | Liver-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 4 isoforms; mature secreted chain 22–523; epitope unknown (UniProt) |
Section 1
Recommended BTD IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published BTD protocol using breast cancer tissue sections (PMC12183389).
| Sample | Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03020) |
| Fixation | Image fixative and duration unreported (datasheet A03020); 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-BTD, 1:100-1:200 (datasheet A03020) |
| 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 | BTD-positive staining in myoepithelial cells of breast (HPA tissue IHC: High). HPA tissue profile: Membranous expression in endothelial cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); compare high-pH retrieval if needed (PMC12183389).
Section 2
What Is the Expected BTD Staining Pattern?
BTD is a secreted protein with no transmembrane segment (UniProt P43251). HPA describes membranous staining in endothelial cells, with high staining in breast myoepithelial cells and cells in kidney glomeruli, and medium staining in hepatocytes (HPA tissue IHC). Interpret that distribution cautiously: HPA rates the tissue staining evidence Uncertain because antibody staining and RNA expression have low consistency, and secreted protein may appear away from its producing cells (HPA tissue IHC).
| Membranous endothelial staining, with stronger signal in the reported breast or kidney cell populations. | This is compatible with the observed HPA profile: membranous endothelial expression, high staining in breast myoepithelial cells and high staining in glomerular cells (HPA tissue IHC). Check cell identity and local tissue architecture before scoring; HPA calls the overall tissue evidence Uncertain (HPA tissue IHC). |
| Predominantly nuclear staining in otherwise intact cells. | A nuclear pattern does not match BTD's secreted annotation or HPA's reported membranous endothelial profile (UniProt P43251; HPA tissue IHC). Treat it as suspect and compare with a control omitting the primary antibody and with the reported positive tissues (general IHC practice; HPA tissue IHC). |
| Strong staining in an unexpected cell population, especially one HPA reports as not detected. | Consider antibody cross-reactivity or endogenous chromogenic detection activity before assigning BTD expression (general IHC practice). HPA reports no detection in adipocytes, bone marrow hematopoietic cells and bronchial respiratory epithelial cells, among other listed populations; its overall rating remains Uncertain (HPA tissue IHC). |
| Diffuse colour across tissue and spaces, with little distinction between structures. | A secreted protein could contribute extracellular signal, so diffuse staining alone does not establish an artefact (UniProt P43251). Broad colour that also appears in a control omitting the primary antibody supports background from the detection workflow; reassess blocking, washing and chromogen development (general IHC practice). |
| No visible signal in breast myoepithelial cells or kidney glomerular cells. | These are high-staining populations in HPA's tissue record, so an absent signal prompts a check of the IHC run, section quality and scoring location (HPA tissue IHC; general IHC practice). Absence does not by itself overturn the HPA pattern or prove a failed assay, because that pattern has Uncertain reliability (HPA tissue IHC). |
💡Expected BTD appearanceA plausible positive slide shows membranous endothelial staining and staining in HPA-reported cells, potentially high in breast myoepithelial or glomerular cells; dominant nuclear colour or uniform colour in primary-omission controls is suspect (HPA tissue IHC; UniProt P43251; general IHC practice).
| Secretion and topology (UniProt P43251) | BTD has a signal peptide at residues 1–21, a mature chain at 22–523 and no transmembrane segment; do not interpret an apparent membrane outline as proof that BTD is membrane anchored (UniProt P43251; HPA tissue IHC). |
| Location of production versus detection (UniProt P43251; HPA tissue IHC) | BTD is secreted, and HPA warns that tissue RNA and protein locations may differ; use the observed staining and cell morphology to interpret a section, rather than treating liver-enhanced RNA as a promise of strongest liver IHC (UniProt P43251; HPA tissue IHC). |
| Reported cell-level contrast (HPA tissue IHC) | HPA reports high staining in breast myoepithelial and kidney glomerular cells, medium staining in hepatocytes, and no detection in several listed cell populations. These are useful comparison points, subject to HPA's Uncertain reliability rating (HPA tissue IHC). |
| Antibody evidence (HPA antibody and tissue IHC records) | The listed antibody HPA052275 has an Uncertain IHC assessment; HPA also rates the tissue pattern Uncertain. A visually convincing slide therefore needs appropriate run controls and cautious interpretation, rather than an assumption of independently verified specificity (HPA antibody and tissue IHC records; general IHC practice). |
| Processing and variants (UniProt P43251) | UniProt lists a cleaved signal peptide, six glycosylation sites and four isoforms. The supplied sources do not locate the antibody epitope or show how these features affect IHC staining; avoid assigning a compartment or intensity change to any one of them (UniProt P43251; HPA antibody record). |
| Situation | Likely cause | Next action |
|---|---|---|
| The HPA-reported high-staining cells are blank. | The comparison population may have been misidentified, or the IHC run may have lost detectable signal (HPA tissue IHC; general IHC practice). | Locate breast myoepithelial cells or kidney glomerular cells by morphology; review section integrity, retrieval, antibody incubation and detection controls as general IHC checks. Do not infer a BTD-specific retrieval requirement from HPA (HPA tissue IHC; general IHC practice). |
| Nuclei dominate the stain. | That pattern conflicts with the secreted annotation and reported membranous endothelial profile; nonspecific staining is a possibility (UniProt P43251; HPA tissue IHC; general IHC practice). | Compare a primary-omission control and a reported positive tissue, then score only anatomically credible signal. HPA's Uncertain rating limits how firmly any single pattern can be declared specific (general IHC practice; HPA tissue IHC). |
| Colour appears broadly, including where no cell pattern is discernible. | Extracellular BTD is possible, while diffuse detection background is also possible; appearance alone cannot distinguish them (UniProt P43251; general IHC practice). | Check the primary-omission control and inspect whether colour tracks extracellular structures or persists throughout the section. Review blocking, washing and chromogen development as general IHC steps (general IHC practice). |
| An HPA-listed negative population stains strongly. | Cross-reactivity or endogenous detection activity could produce colour, and the HPA negative call is itself within an Uncertain tissue profile (general IHC practice; HPA tissue IHC). | Confirm the cell type, compare the primary-omission control, and check the same run's reported positive population. Avoid declaring a new BTD-positive cell type from a single discordant slide (general IHC practice; HPA tissue IHC). |
| Liver stains less strongly than breast or kidney. | That order is compatible with HPA's medium hepatocyte staining versus high staining in breast myoepithelial and glomerular cells; liver-enhanced RNA does not settle protein location (HPA tissue IHC). | Score each named cell population separately and record intensity with the HPA reliability caveat. Do not use liver RNA enrichment alone to call the chromogenic run a failure (HPA tissue IHC). |
| Can IF/ICC establish BTD's subcellular pattern? | HPA labels BTD secreted but provides no main ICC-IF location or cell-line ICC-IF images; the listed antibody has no ICC validation entry (HPA subcellular and antibody records). | Treat IF/ICC localization as unresolved from these sources and use its separate guide for assay design. Keep this page's pattern call tied to the reported tissue IHC observations (HPA subcellular, antibody and tissue IHC records). |
Experiment evidence
IHC & IF images showing BTD staining
A03020 · IHCImmunohistochemistry of paraffin-embedded rat brain using BTD antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM
A03020 · IHCImmunohistochemistry of paraffin-embedded human liver cancer using BTD antibody at dilution of 1:100 .Perform microwave antigen retrieval wi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169814-BTD/tissue/cerebral+cortex#imid_14702928
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169814-BTD/tissue/colon#imid_14702366
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169814-BTD/tissue/liver#imid_14702367
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000169814-BTD/tissue/kidney#imid_14702360
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BTD IHC & IF
🧪Run breast first: myoepithelial cells should stain strongly (HPA: High in breast myoepithelial cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the breast slide, cells outside the myoepithelial layer should show little staining relative to that layer, but should not be assumed absolutely negative (HPA: High in breast myoepithelial cells).
Positive control tissue: Breast (Myoepithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for BTD; derive a cell-line control from the positive tissue's cell type (Myoepithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an irrelevant immunoglobulin control matched to the primary antibody’s host species, isotype, concentration, and clonality; use BTD knockout tissue or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). For chromogenic staining, quench endogenous peroxidase and check background from any biotin-based detection system; for IF, assess tissue autofluorescence with a no-primary control (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the fixative in the selected A03020 paraffin-section caption is unreported (selected A03020 tissue-IHC caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, for rat brain at 1:100; it does not establish a retrieval requirement for breast (selected A03020 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; in breast, assess duct-adjacent extracellular staining cautiously because BTD is secreted (UniProt P43251 subcellular; HPA: High in breast myoepithelial cells).
HPA tissue IHC evidence for BTD
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | Medium | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Stomach | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced BTD IHC Tips
Troubleshoot BTD staining in paraffin sections by checking retrieval, signal location, controls and scoring before interpreting chromogenic IHC (UniProt P43251; HPA tissue IHC).
How should I adjust retrieval when BTD staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page retrieval protocol). If staining remains weak, compare an adjacent section using microwave retrieval in 10 mM PBS, pH 7.2, the condition reported for catalog antibody A03020 in paraffin embedded rat brain; the caption gives no microwave time or temperature (A03020 tissue IHC caption). Keep antibody concentration, detection and imaging conditions matched while comparing retrieval methods (general IHC practice). Stronger staining alone does not establish specificity: evaluate its distribution against control sections and the expected extracellular location of secreted BTD (UniProt P43251 localisation; general IHC practice).
Could fixation explain variable BTD staining between paraffin blocks?
Target specific sensitivity of BTD staining to fixative type or fixation duration is unknown from the supplied evidence; the A03020 paraffin section caption does not state a fixative (A03020 tissue IHC caption). Record each block’s fixative, fixation duration and processing history, then compare sections prepared with the same retrieval and detection conditions (general IHC practice). Test a short retrieval series on matched sections before increasing antibody concentration, because processing differences can change epitope access and background (general IHC practice). Interpret any improvement as an effect under those tested conditions, without assigning it to BTD’s secretion, glycosylation or tissue staining profile (UniProt P43251; HPA tissue IHC).
Where should convincing BTD signal appear, and how should endothelial staining be assessed?
BTD is annotated as secreted into the extracellular space and has no transmembrane segment, so assess extracellular or pericellular staining alongside cell associated signal (UniProt P43251 localisation and topology). HPA describes membranous expression in endothelial cells, but rates its tissue IHC reliability uncertain because staining and RNA expression have low consistency (HPA tissue IHC). Compare vessel associated staining with nearby tissue and a matched control section, taking care to distinguish endothelial boundaries from adjacent extracellular deposits (general IHC practice). A membrane shaped chromogenic outline is insufficient by itself to establish membrane anchoring or intracellular production of BTD (UniProt P43251 topology; general IHC practice).
Could BTD processing or isoforms change what the antibody detects in tissue?
BTD has 4 listed isoforms, and the supplied evidence does not identify which isoforms the catalog antibody recognizes (UniProt P43251 isoforms; supplied antibody evidence). The precursor contains a signal peptide at residues 1–21, while the annotated BTD chain spans 22–523; the epitope position is not supplied (UniProt P43251 processing; supplied antibody evidence). Glycosylation is annotated at 6 sites, which makes epitope accessibility a question to test rather than an established explanation for a staining difference (UniProt P43251 glycosylation; general IHC practice). Compare retrieval conditions and independent specificity controls before attributing discrepant staining to an isoform, cleavage event or glycan effect (general IHC practice).
How can I assess BTD by multiplex IF when this page’s evidence is tissue IHC?
Treat IF as a separate assay requiring its own validation, because the supplied A03020 image documents chromogenic staining of paraffin sections and no ICC/IF images are listed (A03020 tissue IHC caption; HPA subcellular record). Multiplex BTD with a validated marker for the expected cell population, such as an endothelial marker when testing the HPA endothelial pattern, and include single stain controls (HPA tissue IHC; general IF practice). Select a fluorophore channel after measuring tissue autofluorescence, with particular care where it overlaps a dim BTD signal (general IF practice). Because BTD is secreted and lacks a transmembrane segment, first assess accessible extracellular staining; add controlled permeabilisation only if the antibody’s mapped epitope and intended intracellular pool warrant it (UniProt P43251 topology and localisation; general IF practice).
What should I check when BTD staining is diffuse or vessels look uniformly dark?
Run a no primary control and inspect unstained tissue to separate antibody dependent signal from pigment, precipitate and detection background (general IHC practice). In peroxidase based chromogenic IHC, apply an appropriate endogenous peroxidase block and check for residual activity before interpreting DAB deposition (general IHC practice). Adjust blocking, wash stringency and antibody dilution one variable at a time, while keeping exposure to chromogen consistent across compared sections (general IHC practice). Diffuse signal may be compatible with secreted BTD, but staining that follows section edges, damaged areas or every vessel equally needs control based review before being called target specific (UniProt P43251 localisation; general IHC practice).
How should I score BTD staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring, because BTD is secreted and HPA reports both endothelial staining and cell specific tissue signals with uncertain IHC reliability (UniProt P43251 localisation; HPA tissue IHC). For cell associated staining, report the percentage of positive cells or an H-score using fixed intensity thresholds; record extracellular signal separately rather than forcing it into a cellular H-score (general IHC practice). For vessel associated signal, report positive structures per mm² or stained area normalized to total vessel area, using the same segmentation rules across samples (general IHC practice). Normalize counts to evaluable tissue area or the relevant cell population, and exclude folds, necrosis and section edges using predefined criteria (general IHC practice).
How can I distinguish a credible BTD positive pattern from artefact?
Judge a putative positive against BTD’s secreted extracellular location and the sampled tissue’s anatomy, while allowing for cell associated staining that requires independent confirmation (UniProt P43251 localisation; general IHC practice). HPA reports high staining in breast myoepithelial cells and kidney glomerular cells, medium staining in liver hepatocytes, and an uncertain overall tissue IHC reliability rating (HPA tissue IHC). Treat unexpected nuclear dominance, staining confined to section edges or necrosis, and signal present in no primary controls as reasons to investigate artefact (UniProt P43251 localisation; general IHC practice). Where DAB detection is used, check endogenous peroxidase activity and replicate the spatial pattern with appropriate controls before assigning BTD expression (general IHC practice).
Boster reagents
Best BTD / Biotinidase IHC Antibodies
Anti-BTD antibody A03020 has IHC images from paraffin sections of rat brain, human liver cancer and human prostate (catalog: A03020 image captions); its listed reactivity includes Human, Mouse and Rat (catalog: A03020 reactivity).
Real IHC data
Anti-Biotinidase BTD Antibody
Cat # A03020
A03020 will render with its rat brain IHC figure from a paraffin section (catalog: A03020 figure caption). Its other IHC images show paraffin sections of human liver cancer and human prostate (catalog: A03020 image captions).
Which to pick: Choose A03020 for tissue IHC: it is a rabbit polyclonal antibody listed for IHC, with a 1:100–1:200 IHC dilution; its paraffin-section captions specify microwave antigen retrieval in 10 mM PBS at pH 7.2, but do not report the fixative (catalog: A03020 applications, dilution and image captions). For cross-species work, A03020 lists Human, Mouse and Rat reactivity, while its IHC images document human and rat samples only (catalog: A03020 reactivity and image captions). No IF/ICC choice is supported because A03020 has no listed IF application, dilution or image (catalog: A03020 applications, IF dilution and IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P43251 (BTD_HUMAN, Biotinidase).
- Human Protein Atlas. BTD tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. BTD subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. BTD antibody validation summary (1 antibodies).
- CDK9 is up-regulated and associated with prognosis in patients with papillary thyroid carcinoma. Medicine 2022 — PMC8812708.
- Extracellular Vesicle Proteome Analysis Improves Diagnosis of Recurrence in Triple-Negative Breast Cancer. Journal of extracellular vesicles 2025 — PMC12183389.
- Baitong Decoction Ameliorates DSS-Induced Colitis via Modulation of STING and JAK/STAT Pathways. ACS omega 2025 — PMC12593019.
- Liver-originated selective autophagy of BTD by alectinib underlies concurrent hepatotoxicity and dermatotoxicity. Autophagy 2026 — PMC13502023.
- PubMed PMID:7509806 — UniProt-cited evidence.
- PubMed PMID:9530634 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BTD (Biotinidase). Expected localisation Endothelial membranes (HPA tissue IHC); extracellular space (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03020 at 1:100-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Myoepithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03020); 2. Fixation: Image fixative and duration unreported (datasheet A03020); 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-BTD, 1:100-1:200 (datasheet A03020); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Secreted BTD may separate RNA and staining sites (HPA tissue IHC); Isoform / epitope: 4 isoforms; mature secreted chain 22–523; epitope unknown (UniProt); Regulation: Liver-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt P43251, HPA tissue IHC · Uncertain, HPA ICC-IF · Secreted, Datasheet A03020, PMC12183389. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BTD/immunohistochemistry-ihc-guide.