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AK5 / Adenylate kinase isoenzyme 5 · IHC design guide
Design Immunohistochemistry for AK5
Plan AK5 chromogenic IHC around cytoplasmic neuronal staining in brain tissue (HPA tissue IHC). This guide covers FFPE handling, tissue controls, and interpretation of staining.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AK5 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 | Neuronal cytoplasm (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in cortical and hippocampal neurons (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cerebral cortex+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A11378) | |
| Caveat | Presumed off-target staining has been observed (HPA tissue IHC) | |
| Regulation | Brain-enriched RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; verify antibody epitope coverage (UniProt) |
Section 1
Recommended AK5 IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by one published AK5 protocol using colorectal cancer tissue microarrays (PMC8209216).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A11378) |
| Fixation | Image fixative and duration unreported (datasheet A11378); 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-AK5, 1:100-1:300 (datasheet A11378) |
| 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 | AK5-positive staining in neuronal cells of cerebral cortex (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in neuronal cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval). The article assigns its pressure-cooker retrieval step to ITLN1 (PMC8209216).
Section 2
What Is the Expected AK5 Staining Pattern?
AK5 is a cytoplasmic protein with no annotated transmembrane segment or signal peptide (UniProt Q9Y6K8). In paraffin section IHC, expect staining chiefly in neuronal cell cytoplasm, especially in cerebral cortex and hippocampus, where HPA reports high staining (HPA: tissue IHC). HPA rates the tissue IHC evidence Enhanced but notes medium agreement with RNA data and presumed off target binding that was disregarded (HPA: tissue IHC reliability).
| Clear cytoplasmic staining in cerebral cortex or hippocampal neurons, with nuclei comparatively unstained. | This matches the reported neuronal pattern and high staining in both regions (HPA: tissue IHC). Compare cells within the same section: staining should follow neuronal cytoplasm rather than coat every nucleus or the entire tissue. The cytoplasmic location also agrees with UniProt (UniProt Q9Y6K8: subcellular location). |
| Predominantly nuclear, sharply membranous, or extracellular staining replaces the neuronal cytoplasmic pattern. | Treat this as a compartment mismatch requiring validation, rather than evidence that AK5 has moved: UniProt places AK5 in cytoplasm and reports no transmembrane segment or signal peptide (UniProt Q9Y6K8). Review morphology and controls before assigning the stain to AK5 (general IHC practice). |
| Strong staining appears in an unexpected cell population or across many unrelated cell types. | Check the exact tissue and cell type before calling it cross reactivity: HPA also reports high staining in duodenal and small intestinal endocrine cells (HPA: tissue IHC). If the pattern conflicts with the relevant HPA entry, consider nonspecific antibody binding or endogenous chromogenic activity and inspect detection controls (general IHC practice). |
| Low contrast, diffuse colour covers cells and surrounding tissue without a discernible cytoplasmic pattern. | The result cannot establish AK5 localization because the expected neuronal cytoplasm is unresolved (HPA: tissue IHC). Widespread colour can arise from nonspecific detection or residual endogenous activity; evaluate a control omitting primary antibody, blocking, washing, and chromogen development (general IHC practice). |
| No convincing signal appears in cerebral cortex or hippocampal neurons. | That conflicts with HPA's high neuronal staining in these tissues (HPA: tissue IHC). Confirm that the section contains identifiable neurons, then review antibody and detection performance with an appropriate positive control. A blank slide alone cannot distinguish assay failure from a biological difference (general IHC practice). |
💡Expected AK5 appearanceA positive IHC result is distinct cytoplasmic staining in cerebral cortex or hippocampal neurons, reported as high by HPA; dominant nuclear, surface, or indiscriminate tissue staining is a suspect pattern (HPA: tissue IHC; UniProt Q9Y6K8: cytoplasm and topology).
| Cellular compartment | AK5 is cytoplasmic, lacks a transmembrane segment and signal peptide, and is annotated as one chain spanning residues 1–562 (UniProt Q9Y6K8). Score cytoplasmic signal in the relevant cells; do not interpret a cell surface outline as the expected AK5 pattern. |
| Choice of tissue and cell population | UniProt describes AK5 as brain specific, while HPA reports high IHC staining in cortical and hippocampal neurons and in intestinal endocrine cells (UniProt Q9Y6K8: tissue specificity; HPA: tissue IHC). Interpret each observation against its named cell population; brain specificity alone does not invalidate HPA's extra brain staining. |
| Validation and interpretation limit | Both listed antibodies, HPA019128 and HPA057255, have Enhanced IHC status (HPA: antibodies). The tissue profile still carries a medium RNA agreement caveat and records disregarded presumed off target binding (HPA: tissue IHC reliability). Assess unexpected staining with controls instead of treating Enhanced status as proof for every cell. |
| Isoforms | UniProt lists three AK5 isoforms (UniProt Q9Y6K8: isoforms 1, 2 and 3). The supplied records give no antibody epitope or isoform specific staining pattern, so an IHC difference cannot be assigned to a particular isoform from these data. |
| IF/ICC: Where should AK5 appear? | HPA reports mainly cytosolic IF signal, with an additional approved centriolar satellite location (HPA: subcellular ICC-IF). That additional IF finding does not change the expected cytoplasmic readout for paraffin section IHC (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Cortex or hippocampus is blank despite identifiable neurons. | These neurons are reported as high in HPA tissue IHC, so assay performance needs checking (HPA: tissue IHC). | Verify the positive control, antibody and detection steps, and section quality; review retrieval against the antibody's validated IHC procedure (general IHC practice). No AK5 specific fixation sensitivity is established by the supplied sources. |
| Colour is diffuse and obscures cell boundaries. | Nonspecific detection, incomplete blocking or washing, or endogenous activity may raise background (general IHC practice). | Compare a no primary control; check blocking, washing, and chromogen development, then reassess whether neuronal cytoplasm remains distinct (general IHC practice; HPA: tissue IHC pattern). |
| Nuclei or plasma membranes are the dominant positive structures. | The dominant compartment conflicts with cytoplasmic AK5 and its lack of a transmembrane segment (UniProt Q9Y6K8). | Recheck the counterstain and cell boundaries, inspect detection controls, and seek concordant cytoplasmic staining before calling the result AK5 positive (general IHC practice; HPA: tissue IHC). |
| A nonneuronal population stains strongly. | Cell identity matters: HPA reports high endocrine cell staining in duodenum and small intestine, but also flags presumed off target binding in its broader IHC assessment (HPA: tissue IHC). | Identify the tissue and stained cells, compare the matching HPA entry, and investigate discordant staining with controls before assigning specificity (HPA: tissue IHC; general IHC practice). |
| A no primary control develops colour. | Signal without primary antibody points to a detection or endogenous activity contribution, rather than demonstrating AK5 binding (general IHC practice). | Review the detection reagent, endogenous activity blocking, and development time; repeat the control alongside the test section (general IHC practice). |
| IF/ICC shows small puncta as well as cytosolic signal. | HPA reports cytosol as the main IF location and centriolar satellites as an additional location (HPA: subcellular ICC-IF). | Interpret puncta in the IF/ICC context and judge the paraffin section against its neuronal cytoplasmic IHC pattern (HPA: subcellular ICC-IF; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing AK5 staining
A11378 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using AK5 Antibody. The picture on the right is blocked with the synt
A11378-1 · IHCImmunohistochemistry of paraffin-embedded rat brain using AK5 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000154027-AK5/tissue/cerebral+cortex#imid_16694559
HPA · CaudateHuman Protein Atlas IHC, Caudate. Source: https://www.proteinatlas.org/ENSG00000154027-AK5/tissue/basal+ganglia#imid_16694569
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000154027-AK5/tissue/colon#imid_16694493
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000154027-AK5/tissue/liver#imid_16694494
HPA · ICC-IF LHCN-M2Human Protein Atlas immunofluorescence, LHCN-M2, antibody HPA019128; green = AK5, blue = nucleus, red = microtubules.
HPA · ICC-IF LHCN-M2Human Protein Atlas immunofluorescence, LHCN-M2, antibody HPA019128; green = AK5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AK5 IHC & IF
🧪Run cerebral cortex first and look for cytoplasmic staining in neuronal cells (HPA: High in cerebral cortex neuronal cells; UniProt Q9Y6K8: cytoplasm). Run adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the cortex slide, use non-neuronal cells as internal background comparators rather than assuming they are confirmed AK5-negative cells.
Positive control tissue: Cerebral cortex (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AK5 in LHCN-M2, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a host-species-matched isotype control for a monoclonal primary or nonimmune IgG for a polyclonal primary; and a peptide-blocked primary control (A11378 tissue-IHC caption: synthesized-peptide block). For chromogenic brain IHC, quench endogenous peroxidase and inspect the no-primary slide for residual background.
⚠️Feasibility: The supplied evidence reports no AK5-specific fixation window, fixation effect, or antigen-retrieval dependency; the fixative is unreported in the selected paraffin brain caption (A11378 tissue-IHC caption). Optimize antigen retrieval for the paraffin sections, since the supplied evidence does not establish whether frozen sections or IF/ICC are easier (A11378 tissue-IHC caption: paraffin brain). Brain pigment can complicate chromogenic scoring, so compare suspected signal with the no-primary control.
HPA tissue IHC evidence for AK5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 |
|---|---|---|---|---|
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Small intestine | Endocrine cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AK5 IHC Tips
Troubleshoot AK5 staining in paraffin sections by checking retrieval, cell type, cytoplasmic localisation and controls before comparing staining intensity.
What retrieval conditions should I start with for weak AK5 staining in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced antigen retrieval at 95–98 °C for 20 min (page retrieval rule). Allow sections to cool in buffer, then compare the same tissue and antibody dilution across retrieval conditions so changes in staining remain interpretable (standard IHC practice). If staining is weak, test EDTA at pH 9.0 as a fallback alongside the citrate condition, checking for tissue damage and increased background (standard IHC practice). Score neuronal cytoplasmic signal against a no-primary control; AK5 is cytoplasmic and HPA reports cytoplasmic expression in neuronal cells (UniProt Q9Y6K8; HPA tissue IHC).
Can I change fixation to recover weak AK5 staining?
Target-specific sensitivity of AK5 staining to fixation is unknown from the supplied evidence (provided AK5 evidence). The A11378 image shows staining in paraffin-embedded human brain tissue, but its caption does not state the fixative (A11378 tissue-IHC caption). Record fixative, fixation duration and tissue processing for each specimen before comparing chromogenic staining, because these variables can affect antigen access and morphology (standard IHC practice). If a fixation change is necessary, compare matched samples using the same citrate pH 6.0 retrieval, antibody dilution and detection settings (page retrieval rule; standard IHC practice). Interpret any difference as an observed protocol effect, not an established AK5-specific fixation response (provided AK5 evidence).
Which staining pattern should count as AK5 positive in brain sections?
Prioritise cytoplasmic staining in neuronal cells when scoring brain sections: AK5 is assigned to the cytoplasm, and HPA reports neuronal cytoplasmic expression (UniProt Q9Y6K8; HPA tissue IHC). Cerebral cortex and hippocampal neuronal cells are reported as high staining contexts, while caudate neuronal cells are medium (HPA tissue IHC). Check cell outlines and nuclei with a counterstain before assigning diffuse chromogen to a neuronal cytoplasm (standard IHC practice). HPA also reports centriolar satellite localisation from subcellular imaging, but tiny puncta alone are difficult to assign confidently in chromogenic sections (HPA subcellular; standard IHC practice). Compare the suspected pattern with a no-primary control and adjacent tissue morphology (standard IHC practice).
Could isoform choice or epitope masking explain discordant AK5 staining?
AK5 has 3 listed isoforms, and the supplied record does not map this antibody’s epitope onto them (UniProt Q9Y6K8; provided AK5 evidence). Its annotated protein is a 562-amino-acid chain with no signal peptide, transmembrane segment or annotated glycosylation sites (UniProt Q9Y6K8). Those annotations do not establish which isoforms the catalog antibody detects in a paraffin section (UniProt Q9Y6K8; provided AK5 evidence). If staining differs between specimens, compare the same retrieval condition, dilution and detection run before attributing the difference to an isoform (standard IHC practice). The A11378 peptide-blocked image supports competition in that pictured brain section; it does not establish isoform coverage (A11378 tissue-IHC caption).
How should I check an AK5 IHC pattern with multiplex immunofluorescence?
Use IF as a separate localisation check and pair AK5 with a validated marker of neuronal cells, the cell type showing cytoplasmic staining in brain IHC (HPA tissue IHC; standard IF practice). Choose a far-red channel for a dim AK5 signal when tissue autofluorescence is prominent, and inspect single-channel controls before interpreting overlap (standard IF practice). Because AK5 is cytosolic and has no transmembrane segment, use a mild permeabilisation condition compatible with the companion marker to expose intracellular epitopes (UniProt Q9Y6K8; standard IF practice). Keep antibody-only and no-primary controls for spectral bleed-through and background assessment (standard IF practice). HPA subcellular images report mainly cytosolic signal with additional centriolar satellite localisation; evaluate that pattern separately from the chromogenic IHC score (HPA subcellular).
How can I distinguish diffuse AK5 signal from chromogenic background?
Compare a no-primary section with the AK5-stained section under the same detection conditions; shared deposits indicate detection-system background (standard IHC practice). Include a peroxidase block before chromogen development when using peroxidase-based detection, and inspect endogenous pigment separately from DAB precipitate (standard IHC practice). HPA rates its tissue staining Enhanced but reports presumed off-target binding that was disregarded, so an unexpected stained cell population needs independent scrutiny (HPA tissue IHC). The A11378 brain image includes a synthesized-peptide-blocked comparison, providing a control for that depicted antibody application (A11378 tissue-IHC caption). Reduce primary antibody concentration or detection time systematically if background obscures neuronal cytoplasm, while preserving a positive reference section (standard IHC practice; HPA tissue IHC).
What should I measure when comparing AK5 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and neuronal cell population before scoring, because HPA places AK5 staining in neuronal cytoplasm in brain tissue (HPA tissue IHC). For chromogenic sections, record the percentage of positive neuronal cells and an intensity grade of 0–3; their product gives an H-score from 0–300 (standard IHC practice). If counting positive cells instead, report their density per mm² of viable tissue and the total neuronal cells assessed (standard IHC practice). Normalise comparisons to the same cell population, viable area, staining batch and image-analysis thresholds (standard IHC practice). Keep cortex, hippocampus and caudate separate in analysis because their reported neuronal staining levels differ (HPA tissue IHC).
When should I reject an apparent AK5-positive result?
Treat a reproducible neuronal cytoplasmic pattern as the strongest tissue-level match to AK5, particularly in cerebral cortex or hippocampus (UniProt Q9Y6K8; HPA tissue IHC). Question signal confined to nuclei or an unexpected cell population, and compare it with morphology and a no-primary section (UniProt Q9Y6K8; HPA tissue IHC; standard IHC practice). Exclude section edges, folds and necrotic areas from interpretation when chromogen accumulates there (standard IHC practice). Check endogenous peroxidase or pigment when deposits appear in controls or outside identifiable cytoplasm (standard IHC practice). A peptide-blocked A11378 brain image supports competition in that image, while HPA’s noted presumed off-target binding warrants caution about broader specificity (A11378 tissue-IHC caption; HPA tissue IHC).
Boster reagents
Best AK5 / Adenylate kinase isoenzyme 5 IHC Antibodies
Two anti-AK5 antibodies have IHC figures from paraffin-embedded human or rat brain; both list IF, with human and mouse reactivity, and one also lists rat reactivity (catalog; IHC image captions).
Real IHC data
Anti-Adenylate kinase isoenzyme 5 AK5 Antibody
Cat # A11378
Real IHC data
Anti-Adenylate kinase isoenzyme 5 AK5 Antibody
Cat # A11378-1
A11378 lists IHC and IF for human and mouse, with an IHC figure from paraffin-embedded human brain (A11378 catalog; IHC image caption). A11378-1 lists IHC, IF and ICC for human, mouse and rat, with an IHC figure from paraffin-embedded rat brain (A11378-1 catalog; IHC image caption).
Which to pick: For human brain paraffin-section IHC, start with A11378 at the catalog IHC range of 1:100–1:300; its figure shows peptide-blocked staining, but the caption does not report the fixative (A11378 catalog; IHC image caption). For rat brain paraffin-section IHC or an IF/ICC workflow requiring listed rat reactivity, choose A11378-1; its figure uses 1:100 with microwave retrieval in 10 mM PBS, pH 7.2, and its caption does not report the fixative (A11378-1 catalog; IHC image caption). Both are rabbit polyclonals with IF listed, but neither payload includes an IF figure (A11378 and A11378-1 catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6K8 (KAD5_HUMAN, Adenylate kinase isoenzyme 5).
- Human Protein Atlas. AK5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AK5 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the centriolar satellites..
- Human Protein Atlas. AK5 antibody validation summary (2 antibodies).
- Identification of novel DNA hypermethylation of the adenylate kinase 5 promoter in colorectal adenocarcinoma. Scientific reports 2021 — PMC8209216.
- Construction of a Prognostic Model of Prostate Cancer Based on Immune and Metabolic Genes and Experimental Validation of the Gene AK5. Oncology research 2025 — PMC12573196.
- PubMed PMID:10215863 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:16959763 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AK5 (Adenylate kinase isoenzyme 5). Expected localisation Neuronal cytoplasm (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A11378 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebral cortex (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A11378); 2. Fixation: Image fixative and duration unreported (datasheet A11378); 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-AK5, 1:100-1:300 (datasheet A11378); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A11378); Caveat: Presumed off-target staining has been observed (HPA tissue IHC); Isoform / epitope: 3 isoforms; verify antibody epitope coverage (UniProt); Regulation: Brain-enriched RNA expression (HPA tissue RNA). Evidence: UniProt Q9Y6K8, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet A11378, PMC8209216. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AK5/immunohistochemistry-ihc-guide.