AHCY / Adenosylhomocysteinase · IHC design guide

Design Immunohistochemistry for AHCY

Plan paraffin-section IHC for AHCY using its widespread nuclear and cytoplasmic tissue pattern (HPA tissue IHC). The catalog antibody is listed for IHC at 2–5 μg/ml (datasheet A04236-1); compare staining with high-expression kidney tubules and adipocytes where staining was not detected (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for AHCY (IHC for AHCY): expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC), antibody A04236-1, validated IHC image, and IHC protocol steps — Printable **AHCY** IHC protocol sheet — expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC), antibody A04236-1, controls and protocol steps. Open the full AHCY IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

AHCY 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	Nuclear and cytoplasmic staining (HPA tissue IHC)
	Staining pattern	Widespread nuclear and cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A04236-1)
	Positive control ⓘ	Epididymis+4 more · see all
	Negative control ⓘ	Adipose tissue

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Widespread expression limits tissue-negative controls (HPA tissue IHC)
	Regulation	Low tissue specificity (HPA RNA)
	Isoform / epitope	2 isoforms; epitope differences are unspecified (UniProt)

Section 1

Recommended AHCY IHC & IF Protocols

The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: A04236-1). The published IHC protocol below reports AHCY staining in human colorectal samples and mouse colon tissue (PMC9345734).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A04236-1)
Fixation	Image fixative and duration unreported (datasheet A04236-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: EDTA pH 8.0 (datasheet A04236-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04236-1)
Primary antibody	Rabbit anti-AHCY, 2-5 μg/ml (datasheet A04236-1)
Primary incubation	Overnight at 4 °C (datasheet A04236-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A04236-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AHCY-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear and cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A04236-1); the published protocol does not state a retrieval method (PMC9345734).

Section 2

What Is the Expected AHCY Staining Pattern?

AHCY should show nuclear and cytoplasmic staining across many tissues, with strong signal in kidney tubular cells, thyroid glandular cells, and bladder urothelial cells (HPA tissue IHC). HPA rates the tissue profile Approved but reports medium consistency with RNA expression (HPA tissue IHC). AHCY has no transmembrane segment, so a membrane-only pattern needs scrutiny (UniProt P23526 topology).

What am I looking at on my slide?
Kidney tubules show high nuclear and cytoplasmic staining.	This fits the reported high tubular signal and broad nuclear and cytoplasmic profile (HPA tissue IHC). Compare cells within the same section before treating a faint field as a failed stain (standard IHC practice).
Signal forms a sharp cell-surface rim, with little intracellular staining.	A membrane-only pattern is unexpected for AHCY, which lacks a transmembrane segment (UniProt P23526 topology). Check whether the rim persists after control and detection checks; perinuclear signal alone is less decisive because UniProt also lists the endoplasmic reticulum (UniProt P23526 subcellular location).
Adipocytes stain strongly while expected positive cells are faint.	HPA reports AHCY as not detected in adipocytes but high in several other cell types (HPA tissue IHC). Investigate cross-reactivity or endogenous chromogenic activity before assigning that contrast to AHCY (standard IHC practice).
Diffuse color covers cells, stroma, and blank areas.	This does not resolve the reported nuclear and cytoplasmic cell pattern (HPA tissue IHC). Background from detection or inadequate blocking can obscure a genuine intracellular signal; assess a matched negative reagent control (standard IHC practice).
No signal appears in kidney tubules or thyroid glandular cells.	Both are reported as high staining references (HPA tissue IHC). A negative result in one run is inconclusive: review tissue integrity, retrieval, antibody use, and detection with an appropriate positive control (standard IHC practice).

💡Expected AHCY appearanceCall a result positive when identifiable kidney tubular, thyroid glandular, or bladder urothelial cells show high nuclear and cytoplasmic staining (HPA tissue IHC); isolated membrane rims or uniform color outside cells warrant control checks (UniProt P23526 topology; standard IHC practice).

How each factor affects the staining
Which tissue provides a visible reference?	Kidney tubules, epididymal and thyroid glandular cells, testis seminiferous-duct cells, and bladder urothelial cells are reported high; adipocytes are not detected (HPA tissue IHC). The adipocyte result is an observed cell-type finding, not proof that every cell in adipose tissue is negative.
How much weight should the tissue profile carry?	The profile is Approved, with medium consistency between antibody staining and RNA data (HPA tissue IHC). Treat a matching pattern as supportive evidence; resolve an unexpected cell-type pattern with controls rather than assuming the reference is absolute.
What does antibody validation establish?	HPA041225 and HPA044675 are each IHC Approved; HPA044675 is also ICC Supported (HPA antibody validation). These labels support use in the stated applications but do not supply a dilution, retrieval condition, or evidence that both antibodies recognize the same epitope.
Can isoforms or processing predict a different stain?	UniProt lists 2 isoforms, no signal peptide or propeptide, and a chain spanning residues 2–432 (UniProt P23526 isoforms and processing). Without an antibody epitope map, these facts cannot establish which isoform is detected or predict a tissue-specific staining difference.
What is the IF/ICC expectation?	HPA reports supported cytosolic localization in ICC-IF, while tissue IHC is described as nuclear and cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). Use the separate IF/ICC guide for that application; the cell-image result alone does not invalidate nuclear tissue staining.
What can antigen retrieval explain?	Retrieval is a routine variable in paraffin-section IHC (standard IHC practice). No AHCY-specific fixation or retrieval sensitivity is supplied by UniProt or HPA; optimize and assess controls without claiming that fixation caused a particular AHCY result.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A known positive tissue is blank.	An assay or specimen problem is plausible when reported high cells are absent (HPA tissue IHC); this alone does not identify the failed step.	Check section quality, the catalog antibody's IHC-P instructions, retrieval, and chromogenic detection with a run control (standard IHC practice).
The section has diffuse brown background.	Background may arise from blocking, washing, or endogenous detection activity (standard IHC practice). HPA does not attribute a specific AHCY staining pattern to any of these causes.	Inspect a negative reagent control, review blocking and washes, and check endogenous activity controls appropriate to the detection system (standard IHC practice).
Only nuclei stain in tissue IHC.	Nuclear staining is part of the reported pattern, but exclusive nuclear staining differs from HPA's nuclear and cytoplasmic description (HPA tissue IHC).	Compare a reported high reference tissue and review cytoplasmic visibility, detection strength, and control sections before scoring the distribution (HPA tissue IHC; standard IHC practice).
A sharp membrane rim dominates.	AHCY has no transmembrane segment, making a membrane-only assignment questionable (UniProt P23526 topology).	Compare intracellular signal in positive reference cells and inspect negative reagent controls for edge or detection artifacts (HPA tissue IHC; standard IHC practice).
Adipocytes are strongly positive.	That conflicts with the reported not detected adipocyte result, although the HPA tissue profile has only medium RNA consistency (HPA tissue IHC).	Confirm cell identity, compare known positive cells on the run, and assess negative reagent and endogenous activity controls (HPA tissue IHC; standard IHC practice).

Source: UniProt P23526 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AHCY staining

IHC analysis of AHCY using anti-AHCY antibody (A04236-1). AHCY was detected in a paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AHCY Antibody (A04236-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A04236-1 · IHCIHC analysis of AHCY using anti-AHCY antibody (A04236-1). AHCY was detected in a paraffin-embedded section of human breast cancer tissue. He

IHC analysis of AHCY using anti-AHCY antibody (A04236-1). AHCY was detected in a paraffin-embedded section of rat liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AHCY Antibody (A04236-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A04236-1 · IHCIHC analysis of AHCY using anti-AHCY antibody (A04236-1). AHCY was detected in a paraffin-embedded section of rat liver tissue. Heat mediate

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101444-AHCY/tissue/cerebral+cortex#imid_10629709

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101444-AHCY/tissue/colon#imid_10629619

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101444-AHCY/tissue/liver#imid_10629620

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101444-AHCY/tissue/kidney#imid_10629637

HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA044675; green = AHCY, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for AHCY IHC & IF

🧪Run kidney first: cells in tubules should stain for AHCY (HPA: High in kidney tubules). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the kidney slide, cells lacking specific signal should show only counterstain and background chromogen, without assuming that every cell outside the tubules is negative.

Positive control tissue: Epididymis (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AHCY in CACO-2, Hep-G2, KOLF2.1J, with annotated localisation: Cytosol (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control; a host- and clonality-matched isotype control; and AHCY-knockout tissue as a biological negative (standard IHC practice). For chromogenic kidney IHC, quench endogenous peroxidase and check for endogenous biotin if using avidin-biotin detection (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU tissue-IHC caption does not state the fixative (A04236-1 caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for a paraffin section, but its necessity is not established (A04236-1 caption: EDTA retrieval); the supplied evidence does not establish that frozen sections or IF are easier. Kidney tubules can produce endogenous biotin background with avidin-biotin detection (standard IHC practice).

HPA tissue IHC evidence for AHCY

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). 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 →
Testis	Cells in seminiferous ducts	High	Protein (IHC)	HPA →
Thyroid gland	Glandular cells	High	Protein (IHC)	HPA →
Urinary bladder	Urothelial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced AHCY IHC Tips

Use matched paraffin sections and cell level controls to troubleshoot AHCY staining while keeping retrieval, detection, and scoring conditions consistent.

How should I troubleshoot weak AHCY staining after antigen retrieval?

Start with heat mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A04236-1). The selected image used this buffer before overnight incubation with 2 μg/ml antibody at 4°C, but reports no heating duration (caption A04236-1). Use a controlled heating cycle and cooling step, then compare matched sections with identical detection settings so retrieval changes can be judged alongside tissue morphology (standard IHC practice). If staining remains weak, test another retrieval condition as a fallback while retaining EDTA as the reference, and inspect sections for tissue damage (standard IHC practice).

Could fixation explain inconsistent AHCY staining between paraffin blocks?

Target specific fixation sensitivity is unknown for AHCY, and the selected paraffin section caption does not state a fixative (caption A04236-1). Record fixative, fixation duration, delay before fixation, and section thickness, then hold retrieval and staining conditions constant across comparable specimens (standard IHC practice). If batches differ, stain matched control tissue with the same processing history before changing antibody concentration or attributing signal loss to biology (standard IHC practice). Report fixation as an unresolved variable: tissue expression patterns and AHCY's lack of a transmembrane segment provide no direct test of fixation sensitivity (HPA tissue IHC; UniProt P23526 topology).

Should AHCY staining be nuclear, cytoplasmic, or both?

Assess cytoplasmic and nuclear staining separately: tissue IHC reports both, while the supported subcellular location in cell imaging is cytosol (HPA tissue IHC; HPA subcellular). UniProt also lists nucleus, endoplasmic reticulum, and melanosome, with melanosome assignment based on mass spectrometry of isolated fractions (UniProt P23526 subcellular). Compare each compartment with a matched positive control and a section lacking primary antibody; inspect whether staining follows cell boundaries rather than folds or edges (standard IHC practice). A nuclear only result therefore warrants review of morphology, counterstain, and control staining before assigning a biological shift (HPA tissue IHC; standard IHC practice).

Can the IHC antibody distinguish AHCY isoforms or modified epitopes?

AHCY has 2 listed isoforms, but the supplied antibody caption gives no epitope or isoform specificity, so score its staining as AHCY immunoreactivity (UniProt P23526 isoforms; caption A04236-1). The record lists acetylation and phosphorylation among its modifications, yet it does not establish that these alter binding by this antibody (UniProt P23526 modified residues). Review an epitope map or direct validation data before claiming isoform selective or modification selective staining (standard IHC practice). If staining differs between conditions, keep retrieval and detection fixed and use an independent specificity control before interpreting an epitope change (standard IHC practice).

How can I check AHCY localisation by multiplex IF?

For a separate IF experiment, pair AHCY with a validated marker for the expected cell type, such as a glandular cell marker when examining glandular tissue (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low tissue autofluorescence, checking single stained controls for bleed through (standard IF practice). Because AHCY is intracellular and has no transmembrane segment, permeabilise cells to reach intracellular epitopes; the antibody's exact epitope is unreported (UniProt P23526 topology; caption A04236-1; standard IF practice). Compare cytosolic and nuclear signals with cell boundaries and nuclear counterstain, using HPA's supported cytosolic assignment as a reference (HPA subcellular; standard IF practice).

What causes diffuse brown signal in AHCY IHC?

Begin with a section lacking primary antibody and inspect endogenous pigment, precipitate, and staining at tissue edges before calling diffuse DAB signal positive (standard IHC practice). Include a peroxidase blocking step in the chromogenic workflow and check whether residual signal persists in the control; this is general assay practice (standard IHC practice). The selected image used goat serum blocking, an anti rabbit peroxidase secondary, and DAB detection, but gives no evidence that these conditions eliminate background in every tissue (caption A04236-1). If background persists, compare blocking, antibody concentration, washing, and development time one variable at a time on matched sections (standard IHC practice).

How should I score AHCY staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because tissue IHC reports nuclear and cytoplasmic AHCY staining (HPA tissue IHC). For cell level comparisons, report percent positive cells and an H score within the defined population; for spatial comparisons, report positive cell density per mm² of viable tissue (standard IHC practice). Keep exposure, DAB development, counterstain, and positivity thresholds consistent, and have scorers review representative controls before applying the threshold (standard IHC practice). Normalise cell counts to the number of evaluable cells, or density to viable tissue area, while excluding folds and necrotic regions by the same rule across specimens (standard IHC practice).

How do I distinguish true AHCY staining from artefact?

Look for reproducible staining within intact cells, with compartment calls checked against reported nuclear and cytoplasmic tissue staining and supported cytosolic localisation in cell imaging (HPA tissue IHC; HPA subcellular). Use a reported high staining population, such as kidney tubular cells, as a reference and evaluate the same cell type across comparable sections (HPA tissue IHC; standard IHC practice). Be cautious with signal confined to edges, folds, or necrotic areas, or with diffuse brown staining that persists without primary antibody (standard IHC practice). Residual endogenous peroxidase can mimic DAB positivity, so review the peroxidase blocked control before assigning biological meaning to such signal (standard IHC practice).

Boster reagents

Best AHCY / Adenosylhomocysteinase IHC Antibodies

The IHC-validated anti-AHCY antibody has paraffin-section images from human breast cancer and rat liver tissue (catalog IHC captions); no IF image is supplied (catalog).

Real IHC data

Anti-AHCY Antibody ®

Cat # A04236-1

View product →

A04236-1 has IHC images from paraffin sections of human breast cancer and rat liver tissue (catalog IHC captions). Its application list includes IHC but excludes IF/ICC; listed reactivity is human, mouse and rat (catalog).

Which to pick: Choose A04236-1 for paraffin-section IHC in human or rat tissue: its own captions document EDTA retrieval and 2 μg/ml primary antibody (catalog IHC captions). For cross-species planning, mouse is listed as reactive, but the supplied IHC images show only human and rat tissue (catalog); the antibody is polyclonal (catalog dilution record). No IF/ICC-validated SKU is supplied (catalog applications and images), and the fixative used for the paraffin sections is unreported (catalog IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P23526 (SAHH_HUMAN, Adenosylhomocysteinase).
Human Protein Atlas. AHCY tissue IHC expression (reliability: Approved).
Human Protein Atlas. AHCY subcellular location (ICC-IF): Localized to the cytosol..
Human Protein Atlas. AHCY antibody validation summary (2 antibodies).
Screening and cellular validation of prognostic genes regulated by super enhancers in oral squamous cell carcinoma. Bioengineered 2021 — PMC8810015.
Wumei Pill Ameliorates AOM/DSS-Induced Colitis-Associated Colon Cancer through Inhibition of Inflammation and Oxidative Stress by Regulating S-Adenosylhomocysteine Hydrolase- (AHCY-) Mediated Hedgehog Signaling in Mice. Oxidative medicine and cellular longevity 2022 — PMC9345734.
Integrated bulk and single-cell transcriptomics unveil the role of AHCY in cell cycle regulation of endometrial cancer. Frontiers in oncology 2026 — PMC13538023.
Multi-omics characterization identifies AHCY as a prognostic biomarker driving immunometabolic reprogramming in bladder cancer. Translational oncology 2026 — PMC12813242.
PubMed PMID:2596825 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:11780052 — UniProt-cited evidence.