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HIBADH / 3-hydroxyisobutyrate dehydrogenase, mitochondrial · IHC design guide
Design Immunohistochemistry for HIBADH
Plan chromogenic HIBADH IHC in paraffin sections using observed cytoplasmic staining as the tissue reference and mitochondrial localisation as the molecular expectation (HPA tissue IHC; UniProt). Compare high staining in breast glandular cells with undetected staining in oral mucosal squamous cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HIBADH Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic tissue staining; mitochondrial protein (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Oral mucosa+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining varies by cell type, including within epithelia (HPA tissue IHC) | |
| Regulation | No expression regulation specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; mature chain spans aa 37–336 (UniProt) |
Section 1
Recommended HIBADH IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with a published HIBADH staining protocol for bovine testis and epididymis (PMC4489673).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A08760-1) |
| Fixation | Image fixative and duration unreported (datasheet A08760-1); 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-HIBADH, 1:100 - 1:300 (datasheet A08760-1) |
| 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 | HIBADH-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval setting); the article reports citrate retrieval without temperature or duration (PMC4489673).
Section 2
What Is the Expected HIBADH Staining Pattern?
HIBADH is a mitochondrial protein with no annotated transmembrane segment (UniProt P31937: subcellular location and topology). In paraffin-section IHC, expect cytoplasmic staining in many tissues, including high staining in glandular and respiratory epithelial cells (HPA: tissue IHC). HPA rates the tissue pattern Enhanced, while describing antibody staining and RNA expression as only moderately consistent and external verification as pending (HPA: tissue IHC reliability).
| Clear cytoplasmic staining in adrenal or breast glandular cells, or bronchial respiratory epithelial cells. | This agrees with the reported high staining in those cell populations (HPA: tissue IHC). A granular cytoplasmic appearance would be compatible with mitochondrial localisation (UniProt P31937: mitochondrion), but the supplied HPA tissue profile establishes cytoplasmic staining, not a required granule shape. |
| Strong staining appears predominantly in nuclei or along cell membranes. | Treat this as an unexpected compartment and review controls and staining conditions: HPA reports a cytoplasmic tissue pattern (HPA: tissue IHC), and UniProt places HIBADH in mitochondria without an annotated transmembrane segment (UniProt P31937: location and topology). Appearance alone cannot identify the artefact. |
| Strong signal is confined to oral or vaginal squamous epithelium, or to smooth muscle cells. | Those specific cell populations are reported as not detected (HPA: tissue IHC). Suspect nonspecific antibody staining or endogenous chromogen-generating activity, then compare appropriate negative and detection controls (general chromogenic IHC practice). Do not treat an entire tissue as negative when its other cells were not assessed here. |
| Colour spreads across stroma, extracellular spaces, or most cells without clear cellular boundaries. | This is difficult to score as HIBADH: the reported pattern is cellular and cytoplasmic (HPA: tissue IHC). Review background in a matched negative control and the detection-only control; excessive primary concentration, incomplete blocking, or detection background are general IHC possibilities, not established HIBADH-specific effects. |
| No convincing signal appears in a section expected to contain high-staining cells. | First confirm that the relevant cells are present and preserved. Adrenal and breast glandular cells and bronchial respiratory epithelial cells are reported as high (HPA: tissue IHC). A blank result could reflect a failed staining run or unsuitable assay conditions (general IHC practice); it does not establish HIBADH absence. |
💡Expected HIBADH appearanceCall a result concordant when staining is predominantly cytoplasmic in the relevant cells, with strong signal plausible in HPA high populations; isolated strong nuclear, membranous, or HPA not-detected cell staining needs control review (HPA: tissue IHC; UniProt P31937: mitochondrion and topology).
| Cell population and reference intensity | HPA reports high staining in several glandular and epithelial populations, medium in endometrial stromal cells, and no detection in selected squamous epithelial and smooth muscle cells (HPA: tissue IHC). Score the named cell type rather than assigning one intensity to a whole organ. |
| Mitochondrial location and processing | UniProt places HIBADH in mitochondria, annotates a mature chain at residues 37–336, and lists no transmembrane segment (UniProt P31937: location, chain and topology). These annotations guide compartment interpretation; they do not establish an IHC epitope or a particular retrieval condition. |
| Antibody and tissue evidence | Both listed antibodies have Enhanced IHC status (HPA: HPA019522 and HPA021002). The overall tissue reliability description still reports medium agreement with RNA and pending external verification (HPA: tissue IHC). Use these observations as reference patterns, not proof that every sample will match. |
| IF/ICC localisation? | HPA provides no main ICC-IF location and lists no cell lines with ICC-IF images (HPA: subcellular record). Mitochondrial localisation is annotated by UniProt (UniProt P31937: subcellular location), but this payload cannot validate an IF staining pattern or supply an IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Adrenal or breast glandular cells show no chromogenic signal. | The run may have failed, the relevant cells may be absent, or assay conditions may be unsuitable (general IHC practice). These cells are reported high (HPA: tissue IHC). | Check morphology and a known-positive control from the same run, then review primary dilution, retrieval and detection steps against the validated IHC procedure (general IHC practice). Do not infer a HIBADH-specific fixation effect from this result. |
| The stain is mainly nuclear or outlines plasma membranes. | That distribution conflicts with reported cytoplasmic IHC and mitochondrial annotation (HPA: tissue IHC; UniProt P31937: location and topology); the precise cause is undetermined. | Inspect the detection-only control and repeat with an appropriate positive section; compare localisation at the cellular level before scoring (general IHC practice). |
| Oral or vaginal squamous cells stain strongly. | These cell types are reported as not detected (HPA: tissue IHC). Nonspecific binding or endogenous detection activity are possible general IHC causes. | Compare a no-primary control and, for peroxidase detection, verify the endogenous peroxidase blocking step (general chromogenic IHC practice). Reassess only the named squamous cell population. |
| Smooth muscle cells stain as strongly as neighbouring positive cells. | HPA reports no detection in smooth muscle cells (HPA: tissue IHC); broad colour could arise from background or misidentified cell boundaries (general IHC practice). | Check morphology, staining boundaries and matched negative controls. Record the discrepancy by cell type and avoid calling the whole section HIBADH positive solely from this signal. |
| Diffuse chromogen obscures individual cells. | Widespread background prevents comparison with HPA's cytoplasmic cell pattern (HPA: tissue IHC); excessive detection activity or insufficient blocking are general IHC possibilities. | Review negative and detection-only controls, blocking, washes and primary dilution under the validated IHC workflow (general IHC practice). Score only after cellular localisation is distinguishable. |
| IF/ICC gives a pattern that seems inconsistent with IHC. | HPA supplies no ICC-IF images or assigned main subcellular location for this target (HPA: subcellular record), so the supplied evidence cannot resolve the discrepancy. | Interpret the paraffin IHC slide against its tissue and cell controls (HPA: tissue IHC). Consult the separate IF/ICC guide for that application's validation; mitochondrial location alone does not validate its staining pattern (UniProt P31937: mitochondrion). |
Experiment evidence
IHC & IF images showing HIBADH staining
A08760-1 · IHCImmunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue, using HIBADH Antibody. The picture on the right is blocked
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106049-HIBADH/tissue/cerebral+cortex#imid_5207676
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106049-HIBADH/tissue/colon#imid_5207610
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106049-HIBADH/tissue/liver#imid_5207611
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106049-HIBADH/tissue/kidney#imid_5207612
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HIBADH IHC & IF
🧪Run breast first: glandular cells should stain for HIBADH (HPA: High in breast glandular cells). Use oral mucosa as the negative tissue (HPA: Not detected in oral mucosa squamous epithelial cells); on the breast slide, assess background in adjacent non-glandular cells without assuming they are HIBADH-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Oral mucosa (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HIBADH; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class (standard IHC practice). A peptide-block control is supported by the selected antibody’s breast carcinoma image (A08760-1 caption); for chromogenic detection, quench endogenous peroxidase and assess residual tissue background on the no-primary slide (standard IHC practice).
⚠️Feasibility: The selected image uses paraffin-embedded breast carcinoma, but its fixative is unreported (A08760-1 caption); no HIBADH-specific fixation window or antigen-retrieval dependency is reported in the supplied evidence. The evidence does not establish whether frozen sections or IF are easier: HPA lists no ICC-IF images, although HIBADH is mitochondrial (HPA subcellular; UniProt P31937). No breast-specific staining artefact is reported in the supplied evidence.
HPA tissue IHC evidence for HIBADH
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced HIBADH IHC Tips
Troubleshoot HIBADH staining in paraffin sections by checking retrieval, cellular pattern and controls before comparing chromogenic signal across samples.
What retrieval should I try first if HIBADH staining is weak?
Start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen-retrieval setting). Keep section thickness, cooling, antibody concentration and detection conditions consistent while comparing retrieved and unretrieved sections, so a change in signal can be attributed to retrieval (standard IHC practice). If staining remains weak, test a longer or shorter citrate exposure before trying an alternative retrieval buffer as a documented fallback (standard IHC practice). Check whether stronger staining retains a cytoplasmic pattern, since HIBADH is mitochondrial and HPA reports cytoplasmic tissue staining; diffuse nuclear or section-wide color is not convincing rescue (UniProt P31937 subcellular location; HPA tissue IHC profile).
How should I troubleshoot variable HIBADH staining between paraffin blocks?
HIBADH-specific fixation sensitivity is unknown from the supplied evidence; the catalog image identifies paraffin-embedded human breast carcinoma but does not state its fixative (A08760-1 tissue-IHC caption). Record each block’s fixative, fixation duration, processing history and section age, then compare sections using the same retrieval and detection run (standard IHC practice). If archival blocks differ, include a shared control section in each run and interpret differences cautiously until processing effects have been assessed (standard IHC practice). Do not infer fixation tolerance from HPA tissue staining, mitochondrial localization or HIBADH’s annotated modifications; none measures its response to fixation (HPA tissue IHC profile; UniProt P31937 record).
What staining pattern would support mitochondrial HIBADH in tissue?
Look for cytoplasmic staining compatible with a mitochondrial protein, while recognizing that chromogenic IHC generally cannot resolve individual mitochondria reliably (UniProt P31937 subcellular location; HPA tissue IHC profile; standard IHC practice). HIBADH has no annotated transmembrane segment, so a crisp plasma-membrane rim should prompt a check of the antibody, detection reagents and counterstain (UniProt P31937 topology; standard IHC practice). HPA reports high staining in breast glandular cells and respiratory epithelial cells, which can serve as pattern references when those cells are present in a comparison section (HPA tissue IHC). Compare cellular distribution with a negative-reagent control and inspect whether any apparent granular signal stays within cell boundaries rather than following debris (standard IHC practice).
Could processing or modified residues explain discordant HIBADH staining?
The supplied record lists 0 isoforms and a chain spanning residues 37–336, so an apparent tissue pattern difference should not be assigned to a documented alternative isoform (UniProt P31937 processing and isoforms). It also lists acetylation and succinylation at several residues, including alternative modifications at 60 and 76, but gives no antibody epitope or evidence that those modifications alter IHC binding (UniProt P31937 modified residues; supplied antibody evidence). Check the antibody’s disclosed immunogen or epitope information, if available, before proposing a processing-based explanation (standard antibody-validation practice). Compare retrieval and antibody titrations on adjacent sections; the peptide-blocked catalog image supports reagent specificity in that example but does not map the recognized epitope (A08760-1 tissue-IHC caption; standard IHC practice).
How can IF help check an ambiguous HIBADH IHC pattern?
On a separate IF/ICC workflow, multiplex HIBADH with a validated marker for the expected cell type, such as glandular cells in breast tissue, and assess whether signal occupies the same cells (HPA tissue IHC; standard IF practice). Choose fluorophores after measuring tissue autofluorescence in unstained sections, favoring a channel with clear separation from that background rather than judging brightness alone (standard IF practice). Because HIBADH is mitochondrial and has no annotated transmembrane segment, test permeabilisation that gives antibodies access to intracellular epitopes, then verify that cell morphology remains intact (UniProt P31937 subcellular location and topology; standard IF practice). Treat IF colocalization as supporting evidence and compare it with the chromogenic IHC pattern; the supplied HPA record lists no ICC/IF images for this target (HPA subcellular record; standard validation practice).
How do I separate true HIBADH signal from brown background?
Run a section without primary antibody and inspect it before attributing brown deposits to HIBADH (standard chromogenic IHC practice). Include a peroxidase-blocking step in the general detection workflow, then check whether residual color follows blood-rich areas, tissue edges or damaged regions rather than intact cytoplasm (standard chromogenic IHC practice). Titrate the primary antibody and detection reagent while keeping development and counterstain conditions consistent across comparison sections (standard IHC practice). The catalog’s peptide-blocked breast carcinoma image offers a useful specificity comparison for that exact antibody, but peptide competition alone cannot establish that every positive cell or deposit represents HIBADH (A08760-1 tissue-IHC caption; standard antibody-validation practice).
How should I quantify HIBADH staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue compartment before scoring, then apply the same threshold and imaging conditions to every section (standard quantitative IHC practice). For cellular staining, report the percentage of positive cells and, when intensity is reproducible, an H-score formed by summing each intensity category’s percentage times its score (standard quantitative IHC practice). Normalize counts or stained area to evaluable tissue area in mm², or compare H-scores within the same annotated cell population rather than across mixtures of cell types (standard quantitative IHC practice). Exclude folds, edges and necrosis by a prespecified rule, and interpret small differences cautiously because HPA describes medium staining-to-RNA consistency with external verification pending (HPA tissue IHC reliability; standard quantitative IHC practice).
When should I doubt a positive HIBADH IHC result?
A convincing result should show cellular cytoplasmic staining compatible with mitochondrial HIBADH and with the cell population being scored (UniProt P31937 subcellular location; HPA tissue IHC profile). Reassess sharp nuclear or membrane-only staining, signals confined to necrosis or cut edges, and brown deposits that persist without primary antibody (UniProt P31937 topology; standard IHC practice). Compare with relevant HPA patterns: high staining is reported in breast glandular cells, whereas oral-mucosa squamous epithelial cells and smooth muscle cells are reported as not detected (HPA tissue IHC). Also check the peroxidase control for endogenous enzyme activity; HPA’s enhanced reliability designation still notes medium agreement with RNA expression and pending external verification (standard chromogenic IHC practice; HPA tissue IHC reliability).
Boster reagents
Best HIBADH / 3-hydroxyisobutyrate dehydrogenase, mitochondrial IHC Antibodies
A08760-1 is listed for IHC, IF and ICC in human, mouse and rat (catalog: applications/reactivity). Its IHC figure shows paraffin-embedded human breast carcinoma tissue (catalog: IHC image caption).
Real IHC data
Anti-HIBADH Antibody
Cat # A08760-1
A08760-1 will render with an IHC figure of paraffin-embedded human breast carcinoma tissue (catalog: IHC image caption). IF and ICC are listed applications, and human, mouse and rat are listed as reactive species; no IF image is supplied (catalog: applications/reactivity/IF image alts).
Which to pick: For tissue IHC, choose A08760-1 for paraffin sections: its own caption documents human breast carcinoma tissue, but does not report the fixative (catalog: A08760-1 IHC image caption). For IF/ICC, A08760-1 lists both applications and an IF dilution of 1:50 (catalog: A08760-1 applications/IF dilution). For cross-species work, A08760-1 is a rabbit polyclonal listed as reactive with human, mouse and rat; its supplied IHC figure shows human tissue (catalog: A08760-1 host/dilution_raw/reactivity/IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P31937 (3HIDH_HUMAN, 3-hydroxyisobutyrate dehydrogenase, mitochondrial).
- Human Protein Atlas. HIBADH tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HIBADH subcellular location (ICC-IF): Highest expression in SiHa: 108.5 nTPM.
- Human Protein Atlas. HIBADH antibody validation summary (2 antibodies).
- The g.-165 T>C Rather than Methylation Is Associated with Semen Motility in Chinese Holstein Bulls by Regulating the Transcriptional Activity of the HIBADH Gene. PloS one 2015 — PMC4489673.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HIBADH (3-hydroxyisobutyrate dehydrogenase, mitochondrial). Expected localisation Cytoplasmic tissue staining; mitochondrial protein (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08760-1 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Oral mucosa. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A08760-1); 2. Fixation: Image fixative and duration unreported (datasheet A08760-1); 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-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-HIBADH, 1:100 - 1:300 (datasheet A08760-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining varies by cell type, including within epithelia (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain spans aa 37–336 (UniProt); Regulation: No expression regulation specified (UniProt). Evidence: UniProt P31937, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in SiHa: 108.5 nTPM, Datasheet A08760-1, PMC4489673. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HIBADH/immunohistochemistry-ihc-guide.