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ADH5 / Alcohol dehydrogenase class-3 · IHC design guide
Design Immunohistochemistry for ADH5
Plan ADH5 staining in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation, detection, and tissue controls for interpreting chromogenic IHC.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ADH5 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 (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet RP1111) | |
| Positive control | Epididymis+4 more · see all | |
| Negative control | Bone marrow+4 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 | Smooth muscle stains highly; skeletal myocytes are undetected (HPA tissue IHC) | |
| Regulation | No expression regulation annotated (UniProt) | |
| Isoform / epitope | 0 isoforms annotated; no propeptide (UniProt) |
Section 1
Recommended ADH5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet RP1111) is paired with a published ADH5 IHC protocol using Luminal A breast cancer tissue (PMC12000288).
| Sample | Paraffin-embedded human liver cancer tissues; fixative not specified (datasheet RP1111) |
| Fixation | Image fixative and duration unreported (datasheet RP1111); 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 RP1111) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet RP1111) |
| Primary antibody | Rabbit anti-ADH5, 0.5-1μg/ml (datasheet RP1111) |
| Primary incubation | Overnight at 4 °C (datasheet RP1111) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet RP1111) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ADH5-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 for the catalog antibody (datasheet RP1111); consider microwave EDTA pH 9.0 for the published protocol (PMC12000288).
Section 2
What Is the Expected ADH5 Staining Pattern?
ADH5 is a cytoplasmic protein with no transmembrane segment (UniProt P11766). In paraffin-section IHC, expect cytoplasmic staining in hepatocytes, kidney tubular cells, epididymal glandular cells, smooth muscle cells, and cells in seminiferous ducts, all reported as High (HPA tissue IHC). HPA rates the tissue profile Approved, with medium consistency between staining and RNA data; external verification remains pending (HPA tissue IHC).
| Cytoplasmic staining in hepatocytes or kidney tubular cells; the relevant cell populations are reported as High (HPA tissue IHC). | This fits the reported tissue pattern and cytoplasmic location (HPA tissue IHC; UniProt P11766). Judge the signal within identifiable cells and alongside controls, since HPA's Approved rating still carries a medium-consistency caveat (HPA tissue IHC; general IHC practice). |
| Predominantly nuclear or sharply membranous staining, with little cytoplasmic signal (UniProt P11766 location and topology). | That compartment differs from the reported cytoplasmic location and lack of a transmembrane segment (UniProt P11766). Treat it as a pattern requiring artefact and antibody-specificity checks before assigning it to ADH5 (general IHC practice). |
| Strong staining in bone-marrow hematopoietic cells or skeletal-muscle myocytes, both reported as Not detected (HPA tissue IHC). | This conflicts with those specific HPA observations and may reflect cross-reactivity or endogenous detection activity (HPA tissue IHC; general IHC practice). A single discrepant section does not establish a new ADH5 distribution; compare morphology and controls (general IHC practice). |
| Broad chromogen haze across cells, stroma, and empty areas, without a clear cytoplasmic cell pattern (UniProt P11766; general IHC practice). | Poorly localized background cannot be scored as the reported cytoplasmic staining (UniProt P11766; general IHC practice). Inspect a no-primary control and the detection system before comparing apparent intensity among tissues (general IHC practice). |
| No visible staining in hepatocytes or kidney tubular cells, both reported as High (HPA tissue IHC). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
💡Expected ADH5 appearanceA convincing positive shows clear cytoplasmic chromogen in morphologically identifiable High-level cells, such as hepatocytes or kidney tubular cells (HPA tissue IHC; UniProt P11766); isolated nuclear, membranous, or cell-free color is a specificity or background concern (general IHC practice).
| Compartment and topology (UniProt P11766) | UniProt places ADH5 in the cytoplasm and reports no transmembrane segment (UniProt P11766). Use cytoplasmic cellular signal as the localization check; do not interpret a membrane rim alone as the expected pattern (UniProt P11766; general IHC practice). |
| Cell-specific tissue contrast (HPA tissue IHC) | HPA reports High staining in hepatocytes and kidney tubular cells, but Not detected in bone-marrow hematopoietic cells and skeletal-muscle myocytes (HPA tissue IHC). These are cell-specific reference observations, not a claim that every cell in either tissue has the same level (HPA tissue IHC). |
| Strength of IHC evidence (HPA tissue IHC; HPA antibodies) | The tissue profile is Approved, with medium staining–RNA consistency and external verification pending (HPA tissue IHC). HPA044578 and HPA061919 are individually IHC Approved; neither is listed as IHC Enhanced in the supplied antibody record (HPA antibodies). |
| IF/ICC evidence boundary (HPA subcellular; UniProt P11766) | HPA supplies no main ICC-IF location or cell-line images here (HPA subcellular). Cytoplasmic IF would be consistent with UniProt's location, but this record does not establish an observed ICC-IF staining pattern (UniProt P11766; HPA subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| A High-reference tissue has no detectable chromogen (HPA tissue IHC). | The expected cell population may be absent from the section, or the IHC run may lack sensitivity (HPA tissue IHC; general IHC practice). No ADH5-specific fixation or retrieval effect is documented in this payload (HPA tissue IHC). | Confirm the relevant cells by morphology, review the run's positive and reagent controls, then check the catalog antibody's validated IHC-P instructions for retrieval and dilution (general IHC practice). |
| Color is diffuse across the section and obscures cell boundaries (general IHC practice). | Background from detection reagents, inadequate blocking, or excess antibody can mimic widespread staining (general IHC practice). | Compare a no-primary control; review blocking, washes, antibody dilution, and chromogen development against the established IHC workflow (general IHC practice). Score ADH5 only where cellular cytoplasm remains distinguishable (UniProt P11766). |
| The strongest signal is nuclear or outlines cell membranes (UniProt P11766 location and topology). | That pattern does not match UniProt's cytoplasmic location or absence of a transmembrane segment (UniProt P11766); nonspecific staining or detection artefact is possible (general IHC practice). | Inspect no-primary staining and section morphology, then compare a known High cell population on the same run (HPA tissue IHC; general IHC practice). Report the compartment discrepancy if it persists (general IHC practice). |
| Strong signal appears in an HPA Not detected cell population (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible; HPA's Not detected calls apply to specified cells and carry the profile's medium-consistency caveat (HPA tissue IHC; general IHC practice). | Identify the stained cell type, compare a no-primary control, and check the detection system's endogenous-activity control where applicable (general IHC practice). Treat an unresolved mismatch as unconfirmed ADH5 staining (HPA tissue IHC; general IHC practice). |
| A section looks positive, but the signal varies greatly between runs (general IHC practice). | Differences in section handling, retrieval, antibody incubation, or chromogen development can alter IHC appearance (general IHC practice). The supplied sources do not identify an ADH5-specific fixation effect (HPA tissue IHC; UniProt P11766). | Compare matched control tissue across runs and record the IHC conditions before interpreting a biological difference (general IHC practice). Use the HPA cell-specific pattern as a reference, with its stated reliability limit (HPA tissue IHC). |
| Can this IHC pattern serve as an established IF/ICC pattern? (HPA tissue IHC; HPA subcellular) | The supplied HPA subcellular record has no ICC-IF images or reported main location, although UniProt assigns ADH5 to the cytoplasm (HPA subcellular; UniProt P11766). | Treat cytoplasmic IF as a location to test, and assess it with IF-specific controls and the separate IF/ICC guide (UniProt P11766; general IF practice). Do not present the HPA tissue IHC result as IF validation (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing ADH5 staining
RP1111 · IHCIHC analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in paraffin-embedded section of human liver cancer tissues. Heat m
RP1111 · IHCIHC analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in paraffin-embedded section of human liver cancer tissues. Heat m
RP1111 · IFIF analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in immunocytochemical section of A431 cell. Enzyme antigen retrieva
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197894-ADH5/tissue/cerebral+cortex#imid_15138966
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197894-ADH5/tissue/colon#imid_15127900
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197894-ADH5/tissue/liver#imid_15127885
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197894-ADH5/tissue/kidney#imid_15127894
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ADH5 IHC & IF
🧪Run liver first: hepatocytes should stain (HPA: High in hepatocytes); use bone marrow as the negative tissue, where hematopoietic cells are not detected (HPA: Not detected in hematopoietic cells). On the liver slide, unstained neighboring cells can indicate local background, but the supplied HPA row does not establish a specific internal negative cell type (HPA: liver hepatocytes High).
Positive control tissue: Epididymis (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ADH5; 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 a concentration-matched rabbit IgG isotype control appropriate to the primary antibody’s clonality (selected-SKU caption: rabbit anti-ADH5; standard IHC practice). Use ADH5 knockout material, if available, or peptide blocking when a matching immunizing peptide is available as a biological specificity control (standard IHC practice). For liver DAB staining, quench endogenous peroxidase and control endogenous biotin before the streptavidin–biotin detection step (selected-SKU caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish whether ADH5 staining depends on that retrieval condition (selected-SKU caption). Frozen sections and IF/ICC cannot be judged easier from the supplied evidence; liver endogenous peroxidase or biotin may contribute to background with the caption’s SABC/DAB detection (HPA subcellular: no ICC-IF images; selected-SKU caption: SABC with DAB; standard IHC practice).
HPA tissue IHC evidence for ADH5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Testis | Cells in seminiferous ducts | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ADH5 IHC Tips
Troubleshoot ADH5 staining in paraffin sections by checking retrieval, controls, cytoplasmic localisation and cell-specific scoring.
How should I retrieve ADH5 when chromogenic staining is weak?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet RP1111). The RP1111 tissue-IHC image used this treatment before overnight primary-antibody incubation, so compare weak sections with a control processed in the same run (RP1111 tissue-IHC caption). Keep section thickness, heating, cooling and detection conditions consistent while checking whether the weak result repeats; variable processing can obscure a retrieval comparison (standard IHC practice). If staining remains weak, compare retrieval time or an alternative buffer on serial sections, and judge the result by improved cytoplasmic signal without increased background (UniProt P11766 subcellular location; standard IHC practice).
Could fixation explain weak ADH5 staining in my sections?
Target-specific fixation sensitivity is unknown: the RP1111 tissue-IHC caption describes a paraffin section but does not state its fixative (RP1111 tissue-IHC caption). Record the fixative, fixation duration and tissue processing for each specimen, then compare sections prepared under matched conditions (standard IHC practice). A weak result should be checked against a same-run positive control and the stated citrate pH 6 retrieval before changing fixation conditions (HPA: high staining in liver hepatocytes; datasheet RP1111). If a controlled fixation comparison is possible, assess cytoplasmic signal and tissue preservation together; staining differences alone do not establish an ADH5-specific fixation effect (UniProt P11766 subcellular location; standard IHC practice).
Where should ADH5 staining appear in tissue sections?
Score ADH5 primarily as cytoplasmic staining, consistent with its annotated location and lack of a transmembrane segment (UniProt P11766 subcellular location and topology). Use cell identity as a second check: hepatocytes, kidney tubular cells and smooth muscle cells have high tissue-IHC staining in the supplied reference profile (HPA: high in hepatocytes, tubular cells and smooth muscle cells). If signal is mainly nuclear, luminal or restricted to tissue edges, review morphology, retrieval and detection controls before calling it ADH5 (UniProt P11766 subcellular location; standard IHC practice). Record the compartment and cell type separately, because a strong chromogenic deposit in the wrong place does not resolve antibody specificity (standard IHC practice).
How do I assess an unexpected ADH5 staining pattern without a mapped epitope?
The supplied record lists 0 isoforms and no transmembrane segment, so an unexpected compartment cannot be assigned to a documented alternative isoform (UniProt P11766 isoforms and topology). The record also lists modified residues, including phosphoserine at 247 and 324, but gives no antibody epitope or evidence that either modification changes IHC staining (UniProt P11766 modified residues; RP1111 tissue-IHC caption). Compare serial sections under matched retrieval and dilution conditions, using a same-run positive tissue control to test reproducibility (standard IHC practice; HPA: high in liver hepatocytes). Treat a persistent discordant pattern as unresolved until supported by an independent specificity control (standard IHC practice).
How can I check ADH5 localisation with multiplex immunofluorescence?
On a separate IF/ICC guide workflow, pair ADH5 with a validated marker for the expected cell type, such as a hepatocyte marker when examining liver, and check cytoplasmic overlap within identified cells (HPA: high in liver hepatocytes; UniProt P11766 subcellular location). Select a fluorophore outside the strongest tissue autofluorescence and include single-label and no-primary controls to assess bleed-through and background (standard IF practice). Because ADH5 is annotated as cytoplasmic with no transmembrane segment, use permeabilisation appropriate for access to an intracellular epitope after fixation (UniProt P11766 subcellular location and topology; standard IF practice). Optimise IF fixation and permeabilisation independently; the RP1111 paraffin-section caption does not report IF/ICC conditions (RP1111 tissue-IHC caption).
What should I check when ADH5 DAB staining is diffuse?
First compare the section with a no-primary control and inspect whether diffuse deposit persists across compartments or outside intact cells (standard IHC practice). The RP1111 image used 10% goat serum blocking, 1 µg/ml primary antibody overnight at 4°C, and biotin-based DAB detection (RP1111 tissue-IHC caption). Check peroxidase blocking and the detection system as general chromogenic IHC controls, then titrate primary antibody and adjust washing if background remains high (standard IHC practice). Interpret residual cytoplasmic staining against expected cell patterns; hepatocytes stain highly in the supplied tissue profile, whereas skeletal-muscle myocytes were not detected (HPA: high in hepatocytes; HPA: not detected in skeletal-muscle myocytes).
How should I quantify ADH5 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, since ADH5 is annotated as cytoplasmic and reference staining varies by cell type (UniProt P11766 subcellular location; HPA tissue IHC). For a cell-rich region, report the percentage of positive cells and an H-score based on intensity classes; for a spatial question, report positive-cell density per mm² (standard IHC practice). Normalise counts to eligible intact cells or measured viable tissue area, using the same threshold, region rules and staining run across specimens (standard IHC practice). Record necrotic and edge regions separately and compare same-run controls before interpreting a score difference as biological (standard IHC practice).
How do I distinguish convincing ADH5 staining from artefact?
A convincing result places signal in the cytoplasm of intact cells and follows a plausible cell pattern, such as high staining in hepatocytes or kidney tubular cells (UniProt P11766 subcellular location; HPA: high in hepatocytes and kidney tubular cells). Question nuclear or luminal dominance, edge-only staining, and deposits over necrosis, then compare those areas with adjacent preserved tissue (UniProt P11766 subcellular location; standard IHC practice). Persistent DAB signal in a no-primary control can indicate endogenous enzyme activity or detection background rather than antigen-dependent staining (standard IHC practice). Interpret a clean control cautiously because the supplied HPA tissue-IHC reliability is Approved with medium staining–RNA consistency and pending external verification (HPA tissue IHC reliability).
Boster reagents
Best ADH5 / Alcohol dehydrogenase class-3 IHC Antibodies
RP1111 has real IHC data from human liver cancer and mammary cancer paraffin sections and IF data from A431 cells (RP1111 image captions). Human, mouse and rat reactivity is listed (RP1111 catalog).
Real IHC data
Anti-Alcohol dehydrogenase class-3 ADH5 Antibody ®
Cat # RP1111
RP1111 has IHC images from human liver cancer and mammary cancer paraffin sections (RP1111 IHC image captions). It also has an IF image from A431 cells (RP1111 IF image caption).
Which to pick: Choose RP1111 for chromogenic IHC on paraffin sections; its IHC caption documents citrate retrieval at pH 6 for 20 minutes and primary antibody at 1 μg/ml, but does not report the fixative (RP1111 IHC image caption). For IF/ICC, RP1111 has an A431 cell image at 2 μg/ml (RP1111 IF image caption). RP1111 lists human, mouse and rat reactivity, while its supplied IHC images show human tissue only; confirm IHC performance in mouse or rat samples before relying on it (RP1111 catalog; RP1111 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P11766 (ADHX_HUMAN, Alcohol dehydrogenase class-3).
- Human Protein Atlas. ADH5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ADH5 subcellular location (ICC-IF): Highest expression in ASC52telo: 293.0 nTPM.
- Human Protein Atlas. ADH5 antibody validation summary (2 antibodies).
- Elevated ADH5 expression suggested better prognosis in kidney renal clear cell carcinoma (KIRC) and related to immunity through single-cell and bulk RNA-sequencing. BMC urology 2024 — PMC11007970.
- Immunohistochemical Staining Characteristics of Low-Grade Invasive Ductal Carcinoma Using the ADH5 Cocktail (CK5/14, P63, and CK7/18): A Potential Interpretative Pitfall. Diagnostics (Basel, Switzerland) 2023 — PMC10527570.
- ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue. Cell reports 2021 — PMC8640996.
- Genetic associations of plasma proteins and breast cancer identify potential therapeutic drug candidates. Communications biology 2025 — PMC12000288.
- PubMed PMID:2818582 — UniProt-cited evidence.
- PubMed PMID:2679557 — UniProt-cited evidence.
- PubMed PMID:1446828 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ADH5 (Alcohol dehydrogenase class-3). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet RP1111). Validated IHC antibody: RP1111 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Epididymis (Glandular cells); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissues; fixative not specified (datasheet RP1111); 2. Fixation: Image fixative and duration unreported (datasheet RP1111); 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 RP1111); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet RP1111); 8. Primary antibody: Rabbit anti-ADH5, 0.5-1μg/ml (datasheet RP1111); 9. Primary incubation: Overnight at 4 °C (datasheet RP1111). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Smooth muscle stains highly; skeletal myocytes are undetected (HPA tissue IHC); Isoform / epitope: 0 isoforms annotated; no propeptide (UniProt); Regulation: No expression regulation annotated (UniProt). Evidence: UniProt P11766, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in ASC52telo: 293.0 nTPM, Datasheet RP1111, PMC12000288. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ADH5/immunohistochemistry-ihc-guide.