ADH5 / Alcohol dehydrogenase class-3 · IHC design guide

Design Immunohistochemistry for ADH5

Plan chromogenic ADH5 IHC in paraffin sections around the generally cytoplasmic tissue pattern (HPA tissue IHC). The catalog antibody's IHC range is 0.5–1 μg/ml (datasheet RP1111), and liver hepatocytes and kidney tubule cells provide high-staining comparison tissues (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for ADH5 (IHC for ADH5): expected localisation Cytoplasm expected; tissue staining generally cytoplasmic (UniProt; HPA tissue IHC), antibody RP1111, validated IHC image, and IHC protocol steps — Printable **ADH5** IHC protocol sheet — expected localisation Cytoplasm expected; tissue staining generally cytoplasmic (UniProt; HPA tissue IHC), antibody RP1111, controls and protocol steps. Open the full ADH5 IHC guide →

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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	Cytoplasm expected; tissue staining generally cytoplasmic (UniProt; HPA tissue IHC)
	Staining pattern	General cytoplasmic staining across tissue 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	In liver, endogenous peroxidase may mimic DAB staining (HPA tissue IHC; standard IHC practice)
	Regulation	Staining regulation is not established (UniProt)
	Isoform / epitope	No annotated isoforms; cytoplasmic, with no TM segment (UniProt)

Section 1

Recommended ADH5 IHC & IF Protocols

The catalog antibody uses citrate pH 6 heat retrieval (datasheet RP1111). The published ADH5 IHC protocols below cover iBAT (PMC8640996) and breast tissue (PMC12000288).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 citrate pH 6 heat retrieval for the catalog antibody (datasheet RP1111). EDTA pH 9.0 microwave retrieval is a published alternative (PMC12000288).

Section 2

What Is the Expected ADH5 Staining Pattern?

ADH5 is a cytoplasmic protein with no annotated transmembrane segment (UniProt P11766: subcellular location; topology). In paraffin-section IHC, expect cytoplasmic staining in hepatocytes, kidney tubular cells, epididymal glandular cells, smooth muscle cells, and cells in seminiferous ducts; HPA reports these as high (HPA: tissue IHC). HPA rates its tissue pattern Approved, with medium agreement between staining and RNA data and external verification pending (HPA: reliability).

What am I looking at on my slide?
Cytoplasmic signal in hepatocytes or kidney tubular cells (HPA: High in both cell types).	This matches the reported compartment and provides a useful positive reference (UniProt P11766: Cytoplasm; HPA: tissue IHC). Judge signal in the named cells, because a tissue-level positive result does not make every cell in the section positive.
Predominantly nuclear, membranous, or extracellular signal, with little cytoplasmic signal (UniProt P11766: Cytoplasm).	This does not match the annotated location; treat it as a possible staining artefact or antibody specificity issue, not evidence of a new ADH5 location (UniProt P11766: Cytoplasm; general IHC interpretation). Review controls before assigning that pattern to ADH5.
Strong signal in skeletal-muscle myocytes or lymph-node germinal-center cells (HPA: Not detected in those cells).	The reported HPA pattern does not support those cells as strong positives. Consider cross-reactivity or chromogenic detection background, then compare the same run with a reported positive cell population (HPA: tissue IHC; general IHC practice).
Diffuse color across cells and surrounding tissue, without clear cell boundaries (general IHC observation).	A uniform haze is difficult to score as ADH5 because the expected pattern is cellular and cytoplasmic (UniProt P11766: Cytoplasm). Check a no-primary control for background from the detection system and review blocking and wash conditions (general IHC practice).
No convincing cytoplasmic signal in hepatocytes or kidney tubular cells (HPA: High in both).	This conflicts with the reported high IHC levels but does not alone prove ADH5 is absent (HPA: tissue IHC; general IHC interpretation). Check section quality, the positive control, antibody dilution, retrieval conditions, and detection reagents before scoring a negative result (general IHC practice).

💡Expected ADH5 appearanceA convincing positive is cytoplasmic signal in reported high-staining cells, such as hepatocytes or kidney tubular cells (UniProt P11766: Cytoplasm; HPA: High); dominant nuclear, membranous, or cell-free color warrants control review (general IHC interpretation).

How each factor affects the staining
Compartment and topology (UniProt P11766).	ADH5 is annotated in the cytoplasm and has no transmembrane segment; use cytoplasmic staining as the localisation check (UniProt P11766). The record provides no basis for expecting a membrane pattern.
Cell-specific tissue pattern (HPA: tissue IHC).	HPA reports high staining in selected glandular, tubular, hepatocyte, smooth-muscle, and seminiferous-duct cell populations; adipocytes are medium and skeletal-muscle myocytes are not detected (HPA: tissue IHC). Score the specified cells rather than the whole tissue uniformly.
Strength of IHC evidence (HPA: reliability; antibodies).	HPA rates the tissue profile Approved, with medium staining–RNA consistency and external verification pending; HPA044578 and HPA061919 are each IHC Approved (HPA: tissue IHC; antibodies). These ratings support use as references without guaranteeing a result in every specimen.
Can IF/ICC confirm the same pattern? (HPA: subcellular data).	HPA lists no main ICC-IF location and no cell lines with ICC-IF images for ADH5; its tissue IHC pattern cannot establish an IF/ICC result (HPA: subcellular; tissue IHC). Use this page's cytoplasmic expectation as a hypothesis from UniProt, not an IF validation claim (UniProt P11766: Cytoplasm).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A reported high-staining cell population is blank (HPA: tissue IHC).	The run may have insufficient detectable signal; a blank section alone cannot distinguish assay failure from specimen variation (general IHC interpretation).	Check a same-run positive tissue with the named cell population, then review catalog-antibody dilution, retrieval, and detection controls (HPA: tissue IHC; general IHC practice).
Nuclei dominate the signal (UniProt P11766: Cytoplasm).	The compartment conflicts with the annotated cytoplasmic location; nonspecific staining is possible (UniProt P11766; general IHC interpretation).	Compare with a no-primary control and an HPA-reported high cytoplasmic cell population before scoring the nuclear signal as ADH5 (HPA: tissue IHC; general IHC practice).
Color appears in cells HPA reports as not detected (HPA: tissue IHC).	Possible antibody cross-reactivity or detection-system background; HPA's cell-specific finding is a reference, not proof that every specimen is negative (HPA: tissue IHC; general IHC interpretation).	Check the no-primary control, assess cellular localisation, and compare with a reported high-staining population in the same run (UniProt P11766: Cytoplasm; HPA: tissue IHC; general IHC practice).
Diffuse staining obscures cell boundaries (general IHC observation).	Background may arise from detection activity or insufficient blocking or washing (general IHC practice).	Inspect the no-primary control; if it also shows color, review endogenous-enzyme blocking, wash steps, and chromogen development (general chromogenic IHC practice).
Signal is weak in adipocytes or other medium-staining cells (HPA: tissue IHC).	The reference level is medium, so weak signal there is less decisive than loss of signal in an HPA-reported high population (HPA: tissue IHC; general IHC interpretation).	Assess a high-staining reference cell population and keep exposure or chromogen-development conditions consistent across comparisons (HPA: tissue IHC; general IHC practice).
Different sections give conflicting staining intensity (general IHC observation).	Section or run variation may affect comparison; HPA also reports medium staining–RNA consistency and pending external verification (general IHC practice; HPA: reliability).	Compare cell-specific patterns with matched positive and no-primary controls, and document retrieval and detection conditions before assigning biological meaning (HPA: tissue IHC; general IHC practice).

Source: UniProt P11766 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ADH5 staining

IHC analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in paraffin-embedded section of human liver cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-ADH5 Antibody (RP1111) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

RP1111 · IHCIHC analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in paraffin-embedded section of human liver cancer tissues. Heat m

IF analysis of ADH5 using anti-ADH5 antibody (RP1111). ADH5 was detected in immunocytochemical section of A431 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-ADH5 Antibody (RP1111) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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 strongly (HPA: High in liver hepatocytes). Use lymph node germinal center cells as the negative tissue (HPA: Not detected in germinal center cells); HPA does not identify a negative cell type within liver, so do not assume that other cells on the positive slide must be unstained (HPA: liver row).

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, a concentration-matched rabbit IgG isotype control (caption: rabbit primary antibody), and an ADH5 knockout specimen or peptide-blocked primary as a biological specificity control. Quench endogenous peroxidase and assess endogenous biotin in liver when using the caption’s biotinylated secondary and DAB detection (caption: liver tissue, biotinylated secondary, DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected caption does not state the fixative (caption: fixative not stated). Citrate retrieval at pH 6 for 20 minutes was used in the paraffin-section example, but retrieval dependency is not established (caption: citrate retrieval). The evidence does not establish whether frozen sections or IF/ICC are easier; liver endogenous peroxidase and biotin can complicate chromogenic detection, and HPA lists no ICC-IF images for ADH5 (HPA: subcellular record; caption: biotinylated secondary and DAB).

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

Use cytoplasmic staining, matched controls and consistent processing to assess ADH5 in paraffin sections (UniProt P11766; HPA tissue IHC).

How should I optimize retrieval if ADH5 staining is weak?

Start with heat-mediated citrate antigen retrieval at pH 6 for 20 minutes (datasheet RP1111). The matched paraffin-section image used this treatment before overnight incubation with 1 μg/mL primary antibody at 4°C (datasheet RP1111). If signal is weak, compare retrieval times on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Include a liver section as a positive control and assess hepatocyte cytoplasm, where ADH5 staining is reported as high (HPA: liver hepatocytes; UniProt P11766: cytoplasm). Excessive retrieval can damage morphology or increase nonspecific staining, so choose the mildest condition that gives interpretable signal (standard IHC practice).

Could fixation explain weak or patchy ADH5 staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet RP1111). Record fixative, fixation duration and processing history for each specimen, then compare sections processed alike (standard IHC practice). If staining varies with processing, test adjacent sections using the same citrate pH 6 retrieval, antibody concentration and chromogenic development before attributing the difference to ADH5 abundance (datasheet RP1111; standard IHC practice). Judge preservation using tissue morphology and an appropriate positive control on the same run (standard IHC practice). Neither the reported cytoplasmic location nor the annotated modifications establish a fixation effect (UniProt P11766: cytoplasm and modified residues).

Where should convincing ADH5 staining appear in a tissue section?

Expect predominantly cytoplasmic staining: ADH5 is annotated in the cytoplasm and has no transmembrane segment (UniProt P11766). The tissue IHC profile also describes general cytoplasmic expression, with high staining in liver hepatocytes and kidney tubule cells (HPA: tissue IHC). Compare the compartment within identified cells, because diffuse brown precipitate over lumina or damaged tissue is difficult to assign to cytoplasm (standard IHC practice). Use a counterstain to check cell boundaries and nuclear morphology, and inspect an antibody-omission control for detection-system signal (standard IHC practice). A strong membrane-restricted or nuclear-only pattern needs independent validation before assignment to ADH5 (UniProt P11766: cytoplasm; standard IHC practice).

How do I evaluate an unexpected staining pattern when the antibody epitope is unclear?

The supplied record lists 0 isoforms and a chain spanning residues 2–374, but gives no antibody epitope (UniProt P11766; datasheet RP1111). Do not explain an unexpected compartment or cell pattern by proposing an unlisted isoform (UniProt P11766: isoforms and cytoplasm). Ask for the immunogen or epitope location, then assess whether that region could be altered by processing without assuming that it is (standard IHC practice). Annotated modifications include acetylation, succinylation and phosphorylation; their presence alone does not establish interference with this antibody (UniProt P11766: modified residues). Compare the pattern with a second independently validated antibody or another orthogonal measure where available (standard IHC practice).

How should I assess ADH5 by multiplex IF alongside this IHC guide?

Treat IF as a separate assay requiring its own validation; the supplied product image documents paraffin-section chromogenic IHC (datasheet RP1111). Pair ADH5 with a marker identifying the expected cell population, such as hepatocytes in liver, and check for cytoplasmic signal within those cells (HPA: liver hepatocytes; UniProt P11766: cytoplasm). Select spectrally separated fluorophores and place the weaker target signal in a channel with lower tissue autofluorescence, checking single-stain and unstained controls (standard IF practice). Because ADH5 is cytoplasmic and lacks a transmembrane segment, evaluate permeabilisation for access to the intracellular epitope after fixation (UniProt P11766; standard IF practice). Optimize fixation and permeabilisation empirically; no target-specific IF conditions are supplied (standard IF practice).

What should I check when DAB background obscures ADH5 staining?

First compare the stained section with a primary-antibody omission control to locate background from secondary reagent or chromogen development (standard IHC practice). In the selected paraffin-section image, the tissue received 10% goat serum blocking, a biotinylated goat anti-rabbit secondary and SABC with DAB (datasheet RP1111). Block endogenous peroxidase for a peroxidase-based DAB workflow, and assess endogenous biotin when using an avidin–biotin detection system (standard IHC practice). If background remains, shorten chromogen development or titrate the primary while retaining the documented 1 μg/mL condition as a comparison (datasheet RP1111; standard IHC practice). Evaluate cytoplasmic signal against intact tissue morphology rather than diffuse surface precipitate (UniProt P11766: cytoplasm; standard IHC practice).

How should I score ADH5 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and region before scoring, then record cytoplasmic intensity and the percentage of positive cells to calculate an H-score (UniProt P11766: cytoplasm; standard IHC practice). For a cell-count endpoint, report positive cells per mm² of viable tissue and document how the area was measured (standard IHC practice). Normalize comparisons to the same cell type, viable tissue area, section-processing batch and chromogen exposure rather than total section area alone (standard IHC practice). Keep thresholds fixed using controls and review representative fields across each section (standard IHC practice). Interpret differences cautiously because the HPA IHC profile has medium agreement with RNA data and awaits external verification (HPA: reliability description).

How can I distinguish true ADH5 staining from artefact?

A credible signal is cytoplasmic within identifiable cells and matches the expected tissue pattern, such as hepatocytes or kidney tubule cells (UniProt P11766: cytoplasm; HPA: tissue IHC). Be cautious with nuclear-only or membrane-rim staining, since the supplied record places ADH5 in cytoplasm and lists no transmembrane segment (UniProt P11766). Exclude section edges, necrotic regions and displaced DAB precipitate when judging positive cells (standard IHC practice). Compare an antibody-omission control to detect staining from endogenous peroxidase or the biotin-based detection system used in the selected image (datasheet RP1111; standard IHC practice). Confirm surprising patterns with an independent reagent or orthogonal assay before assigning biological meaning (standard IHC practice).

Boster reagents

Best ADH5 / Alcohol dehydrogenase class-3 IHC Antibodies

RP1111 has real ADH5 IHC data from paraffin-embedded human liver cancer tissue and IF/ICC data from A431 cells (RP1111 image captions).

Real IHC data

Anti-Alcohol dehydrogenase class-3 ADH5 Antibody ®

Cat # RP1111

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RP1111 will render with its human liver cancer paraffin-section IHC figure (RP1111 IHC image caption). Additional captions document human mammary cancer paraffin sections and A431 IF/ICC; the catalog lists Human, Mouse, and Rat reactivity (RP1111 IHC and IF image captions; RP1111 catalog reactivity).

Which to pick: Choose RP1111 for tissue IHC: its own figure documents staining in human liver cancer paraffin sections after citrate retrieval at pH 6 for 20 minutes (RP1111 IHC image caption). For IF/ICC, RP1111 has an A431 cell image using 2 μg/mL antibody (RP1111 IF image caption). RP1111 lists Human, Mouse, and Rat reactivity, but the supplied tissue images show human samples; the IHC caption does not report the fixative (RP1111 catalog reactivity; 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.