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KIF5A / Kinesin heavy chain isoform 5A · IHC design guide
Design Immunohistochemistry for KIF5A
Plan KIF5A paraffin IHC using cerebral cortex or hippocampus neuronal cells as positive controls (HPA tissue IHC). This guide covers cytoplasmic staining in neuronal subsets and the possibility of off-target signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
KIF5A 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); perinuclear enrichment expected (UniProt) | |
| Staining pattern | Cytoplasmic staining in subsets of neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cerebral cortex+2 more · see all | |
| Negative control | Adipose tissue+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 | Presumed off-target staining can complicate scoring (HPA tissue IHC) | |
| Regulation | Enriched in neuronal subsets (UniProt) | |
| Isoform / epitope | No isoforms recorded; no extracellular segment (UniProt) |
Section 1
Recommended KIF5A IHC & IF Protocols
The catalog antibody protocol is followed by published KIF5A IHC methods for bladder, liver and canine nervous tissue (PMC6636440; PMC9853384; PMC4864272).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A03635); 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-KIF5A, 5 μg/mL (datasheet A03635) |
| 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 | KIF5A-positive staining in neuronal cells of cerebral cortex (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression mainly in subsets of neuronal cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (this page’s antigen-retrieval guidance); the liver study specifies a hot water bath without a buffer (PMC9853384).
Section 2
What Is the Expected KIF5A Staining Pattern?
KIF5A should appear chiefly in the cytoplasm of subsets of neurons, with concentration around the cell body and perinuclear region (HPA tissue IHC; UniProt Q12840). It has no transmembrane segment (UniProt Q12840 topology). HPA rates the tissue IHC pattern Enhanced, while reporting medium consistency with RNA expression and presumed off-target staining that was disregarded (HPA tissue IHC).
| Cytoplasmic staining in subsets of cerebral cortex or hippocampal neurons. | This matches the reported neuronal pattern; both tissues show medium staining in neuronal cells (HPA tissue IHC). Assess the stained cells and their cytoplasmic distribution together: UniProt places KIF5A in the neuronal cell body, particularly around the nucleus (UniProt Q12840). |
| Predominantly nuclear staining in a paraffin section, with little neuronal cytoplasmic signal. | Treat this as an IHC pattern mismatch and investigate nonspecific staining or detection artefact before scoring it as KIF5A (HPA tissue IHC; UniProt Q12840). HPA separately reports approved nucleoplasmic and cytosolic ICC-IF localisation; that ICC-IF observation does not establish a nuclear IHC tissue pattern (HPA subcellular ICC-IF). |
| Strong staining across cell types reported as negative in the same tissue. | Check cell identity before calling the tissue positive. For example, HPA reports adipocytes in adipose tissue and hematopoietic cells in bone marrow as not detected (HPA tissue IHC). Unexpected staining may reflect cross-reactivity or endogenous detection activity; compare it with a negative reagent control (general IHC practice). |
| Diffuse chromogen over cells and surrounding tissue, obscuring cytoplasmic borders. | Background that lacks the reported subset-of-neurons pattern cannot establish KIF5A localisation (HPA tissue IHC). Review reagent controls, blocking, washes, antibody concentration and development time as general IHC troubleshooting steps; HPA reports presumed off-target binding in its tissue assessment (HPA tissue IHC; general IHC practice). |
| No neuronal signal in cerebral cortex or hippocampus. | First confirm that the section contains identifiable neuronal cells and that detection worked: HPA reports medium neuronal staining in both tissues (HPA tissue IHC). A blank result is inconclusive until the IHC workflow and antibody performance are checked (general IHC practice); it does not by itself overturn the reported tissue pattern. |
💡Expected KIF5A appearanceCall a positive IHC result when subsets of cerebral cortex or hippocampal neurons show medium cytoplasmic staining, often concentrated around the cell body or nucleus (HPA tissue IHC; UniProt Q12840); widespread staining of HPA-negative cell types or dominant nuclear-only staining warrants investigation (HPA tissue IHC; general IHC practice).
| Choice of positive and comparison tissues | Cerebral cortex and hippocampus provide reported medium neuronal staining; cerebellar Purkinje cells and caudate neurons are reported low (HPA tissue IHC). A weak result in a low-level tissue is less decisive than a blank result in a reported medium-level neuronal population (HPA tissue IHC; general IHC practice). |
| Antibody validation and specificity | HPA004469 is rated IHC Enhanced, meaning its pattern was supported by independent antibody staining or orthogonal data (HPA antibodies). The tissue assessment also notes presumed off-target binding, so confirm cell type and compartment and review controls before accepting unexpected staining (HPA tissue IHC; general IHC practice). |
| Cellular distribution and topology | KIF5A is a cytoplasmic, cytoskeleton-associated motor concentrated in neuronal perikarya, especially perinuclearly, and lacks a transmembrane segment (UniProt Q12840). Use this distribution to interpret tissue staining; the record supplies no basis for requiring a membrane-only IHC pattern (UniProt Q12840). |
| Antigen retrieval in paraffin sections | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Q: Does ICC-IF localisation define the IHC result? | A: No. HPA approves nucleoplasmic and cytosolic ICC-IF localisation, with additional locations reported, while tissue IHC mainly shows cytoplasmic staining in neuronal subsets (HPA subcellular ICC-IF; HPA tissue IHC). Interpret an IF image in its own assay context; no IF protocol is specified here. |
| Situation | Likely cause | Next action |
|---|---|---|
| Neurons are unstained in a cerebral cortex or hippocampus section. | The expected medium neuronal signal may be missed because of a workflow or antibody performance problem (HPA tissue IHC; general IHC practice). | Verify neuronal morphology, check the positive control and detection reagents, then review the antibody's validated IHC conditions and retrieval settings (general IHC practice). |
| Only nuclei stain in a paraffin section. | The pattern conflicts with the mainly cytoplasmic neuronal tissue pattern; nuclear ICC-IF localisation does not validate nuclear-only IHC (HPA tissue IHC; HPA subcellular ICC-IF). | Compare with a reagent control, inspect counterstain and chromogen localisation, and reassess specificity before scoring cells as positive (general IHC practice). |
| Adipocytes in adipose tissue or marrow hematopoietic cells stain strongly. | These cell populations are listed as not detected; HPA also notes presumed off-target binding in its tissue assessment (HPA tissue IHC). | Confirm cell identity and compare staining with a negative reagent control; investigate antibody specificity and endogenous detection activity (general IHC practice). |
| Background covers most of the section. | Nonselective chromogen deposition can obscure the reported neuronal cytoplasmic pattern (HPA tissue IHC; general IHC practice). | Review blocking, washes, reagent concentration and chromogen development; use a control lacking primary antibody to assess detection background (general IHC practice). |
| Cerebellar Purkinje cells appear faint or absent. | HPA reports low staining in Purkinje cells, so this tissue offers a less decisive positive comparison than cortex or hippocampus (HPA tissue IHC). | Compare with reported medium-level neuronal staining in cerebral cortex or hippocampus before judging assay performance (HPA tissue IHC; general IHC practice). |
| Enterocytes stain despite the chiefly neuronal summary. | HPA reports medium duodenal enterocyte staining as well as mainly neuronal cytoplasmic expression; the summary does not make every non-neuronal signal false (HPA tissue IHC). | Check tissue and cell identity, localisation and controls. Record the enterocyte result separately from the neuronal pattern instead of dismissing it solely by cell type (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing KIF5A staining
A03635 · IHCImmunohistochemistry of KIF5 in mouse brain tissue with KIF5 antibody at 5 μg/ml.
A03635 · IFImmunocytochemistry of KIF5 in K562 cells with KIF5 antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000155980-KIF5A/tissue/cerebral+cortex#imid_1038
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000155980-KIF5A/tissue/cerebral+cortex#imid_20421115
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000155980-KIF5A/tissue/colon#imid_20418097
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000155980-KIF5A/tissue/liver#imid_20418106
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA073448; green = KIF5A, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA073448; green = KIF5A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for KIF5A IHC & IF
🧪Run cerebral cortex first; neuronal cells should stain at the medium level (HPA: cerebral cortex, neuronal cells). Use adipose tissue as the negative tissue (HPA: adipocytes not detected); non-neuronal cells on the cortex slide should provide a background reference, with staining judged against the neuronal cells identified by HPA (HPA: cerebral cortex, neuronal cells).
Positive control tissue: Cerebral cortex (Neuronal cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show KIF5A in Hep-G2, SH-SY5Y, U2OS, Sperm, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and irrelevant IgG controls matched to the primary antibody’s host species and, for a monoclonal antibody, its isotype; use KIF5A-knockout tissue as a biological negative where available (standard IHC practice). Block endogenous peroxidase in cortex sections and distinguish neuronal staining from endogenous pigment when scoring chromogenic signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependence are unreported in the supplied evidence; the 5 μg/ml mouse brain IHC caption for A03635 does not report a fixative (A03635 caption). The supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC, although HPA provides ICC-IF images (HPA: KIF5A subcellular). Brain pigment can complicate chromogenic scoring (standard IHC practice).
HPA tissue IHC evidence for KIF5A
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 | Medium | Protein (IHC) | HPA → |
| Duodenum | Enterocytes | Medium | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced KIF5A IHC Tips
Troubleshoot KIF5A chromogenic IHC in paraffin sections using neuronal staining patterns, controlled processing changes, and compartment aware scoring.
How should I adjust retrieval when neuronal KIF5A staining is weak?
Begin with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Include a neuronal positive control because KIF5A is enriched in subsets of CNS neurons (UniProt Q12840 tissue specificity). If staining remains weak, pilot an alkaline buffer on adjacent sections while keeping antibody incubation and chromogen development constant (standard IHC practice). Compare cytoplasmic and perinuclear signal with a no primary control, and reject conditions that increase diffuse background or obscure cell boundaries (UniProt Q12840 subcellular; standard IHC practice).
Could fixation explain absent KIF5A staining in paraffin sections?
Target specific fixation sensitivity is unknown: the selected mouse brain IHC caption gives 5 µg/ml antibody but does not state the fixative (selected SKU A03635 caption). Record fixation and processing conditions for each section before attributing a weak result to the antibody (standard IHC practice). Compare adjacent sections processed under documented conditions using the same citrate pH 6.0 retrieval and identical detection settings (page retrieval rule; standard IHC practice). Check tissue preservation and a no primary control, then judge signal in neuronal cytoplasm and perinuclear regions (UniProt Q12840 subcellular; standard IHC practice).
Which cellular staining pattern is credible for KIF5A IHC?
Expect predominantly neuronal cytoplasmic staining, with possible concentration around the cell body and perinuclear region (HPA tissue IHC; UniProt Q12840 subcellular). KIF5A associates with the cytoskeleton and supports axonal transport, so evaluate processes in anatomical context rather than scoring every fine profile as a separate positive cell (UniProt Q12840 subcellular and function; standard IHC practice). HPA reports mainly nucleoplasmic and cytosolic localisation in its subcellular dataset, which can complicate interpretation of nuclear signal (HPA subcellular). Compare nuclear staining with the no primary control and neuronal morphology before calling it specific in chromogenic sections (standard IHC practice).
How can epitope uncertainty affect KIF5A IHC interpretation?
The supplied record lists 0 isoforms and places the kinesin motor domain at residues 9–327; it does not identify this antibody’s epitope (UniProt Q12840 record). KIF5A has no transmembrane segment or signal peptide, so interpret staining primarily within cellular compartments rather than as a surface rim (UniProt Q12840 topology and processing). Document the antibody’s stated immunogen, if available, before comparing another reagent or interpreting discordant staining (standard IHC practice). Acetylation at residue 2 and phosphorylation at 397 are annotated, but their effects on this antibody’s IHC binding are unknown (UniProt Q12840 modified residues).
How should I compare a multiplex IF result with chromogenic KIF5A IHC?
Use an expected neuronal cell marker in multiplex IF to test whether fluorescent KIF5A signal belongs to neurons identified in the IHC section (UniProt Q12840 tissue specificity; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence and include single stain controls to assess channel bleed through (standard IF practice). KIF5A has no transmembrane segment and is reported in cytoplasmic and perinuclear compartments, so use permeabilisation appropriate for an intracellular epitope (UniProt Q12840 topology and subcellular). The selected mouse brain IHC caption reports 5 µg/ml but no IF conditions; optimise IF independently and compare cellular patterns rather than equating signal intensity across detection methods (selected SKU A03635 caption; standard IF practice).
What should I check when the DAB signal obscures neuronal staining?
Examine a no primary control first to identify detection background, and include a peroxidase block before DAB development as part of the chromogenic workflow (standard IHC practice). Titrate the IHC validated antibody and shorten chromogen development systematically while preserving the same retrieval conditions across comparison sections (standard IHC practice). Check for staining concentrated at section edges, folds, or damaged areas before treating those deposits as KIF5A signal (standard IHC practice). Genuine tissue signal should remain interpretable in neuronal cytoplasm, while HPA reports presumed off target binding in its tissue staining assessment (UniProt Q12840 subcellular; HPA reliability description).
How should I quantify KIF5A staining across neuronal regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define neuronal regions and cell inclusion criteria before scoring because KIF5A is enriched in subsets of CNS neurons (UniProt Q12840 tissue specificity; standard IHC practice). Report either an H score for cytoplasmic intensity, the percentage of positive neurons, or positive neuronal profiles per mm², with the chosen threshold stated (standard IHC practice). Normalise counts to eligible neurons or sampled tissue area, and keep retrieval, antibody exposure, imaging, and DAB development consistent between sections (standard IHC practice). Record perinuclear signal separately if relevant, and avoid treating HPA’s medium cerebral cortex staining as a universal intensity standard (UniProt Q12840 subcellular; HPA tissue IHC).
When is apparent KIF5A positivity likely to be artefactual?
A credible result places signal in neuronal cytoplasm or perinuclear regions within an appropriate tissue context (HPA tissue IHC; UniProt Q12840 subcellular). Question strong signal confined to unrelated cell types, section edges, folds, or necrotic areas, and compare those sites with a no primary control (standard IHC practice). Persistent chromogen without primary antibody can indicate endogenous enzyme activity or detection background rather than KIF5A (standard IHC practice). Interpret isolated nuclear staining cautiously: HPA reports nucleoplasmic localisation in its subcellular dataset, while its tissue IHC profile is mainly cytoplasmic and notes presumed off target binding (HPA subcellular; HPA tissue IHC and reliability description).
Boster reagents
Best KIF5A / Kinesin heavy chain isoform 5A IHC Antibodies
The catalog antibody has a mouse brain IHC image and K562 ICC and IF images (catalog image captions). Human, mouse, and rat reactivity is listed; KIF5A isoform specificity is unreported (catalog: reactivity and title).
Real IHC data
Anti-KIF5 Antibody
Cat # A03635
A03635 will render with its mouse brain IHC image at 5 μg/mL (A03635 IHC image caption). Its ICC and IF images show K562 cells, and an IF image shows mouse brain tissue (A03635 IF image captions).
Which to pick: Choose A03635 for tissue IHC because its own image shows staining in mouse brain; section processing and fixative are unreported (A03635 IHC image caption). Choose the same SKU for IF/ICC based on its K562 and mouse brain images (A03635 IF image captions). It is a rabbit antibody listed for human, mouse, and rat, but the captions do not demonstrate IHC in all three species or establish KIF5A isoform specificity (catalog: host, reactivity, title; A03635 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q12840 (KIF5A_HUMAN, Kinesin heavy chain isoform 5A).
- Human Protein Atlas. KIF5A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. KIF5A subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to the plasma membrane, mitotic spindle, mid piece and principal piece..
- Human Protein Atlas. KIF5A antibody validation summary (2 antibodies).
- KIF5A Promotes Bladder Cancer Proliferation In Vitro and In Vivo. Disease markers 2019 — PMC6636440.
- Axonal transport deficit in a KIF5A( -/- ) mouse model. Neurogenetics 2012 — PMC3332386.
- KIF5A upregulation in hepatocellular carcinoma: A novel prognostic biomarker associated with unique tumor microenvironment status. Frontiers in oncology 2022 — PMC9853384.
- Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis. Brain and behavior 2016 — PMC4864272.
- PubMed PMID:7514426 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:19413330 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for KIF5A (Kinesin heavy chain isoform 5A). Expected localisation Neuronal cytoplasm (HPA tissue IHC); perinuclear enrichment expected (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03635 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Cerebral cortex (Neuronal cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A03635); 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-KIF5A, 5 μg/mL (datasheet A03635); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining can complicate scoring (HPA tissue IHC); Isoform / epitope: No isoforms recorded; no extracellular segment (UniProt); Regulation: Enriched in neuronal subsets (UniProt). Evidence: UniProt Q12840, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A03635, PMC6636440, PMC9853384. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KIF5A/immunohistochemistry-ihc-guide.