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AKT3 / RAC-gamma serine/threonine-protein kinase · IHC design guide
Design Immunohistochemistry for AKT3
Plan chromogenic IHC-P for AKT3 using cytoplasmic and nuclear staining as the expected readout (HPA tissue IHC). Compare high-staining caudate neurons with salivary glandular cells where staining was not detected (HPA tissue IHC), and titrate the IHC-validated antibody at 1:50–1:100 (datasheet: A00520-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AKT3 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic and nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Variable cytoplasmic and nuclear staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Salivary gland+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Use consistently formalin-fixed, paraffin-embedded sections (datasheet: A00520-1 IHC-P) (selected-SKU IHC image A00520-1) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Staining intensity varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended AKT3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published AKT3 staining parameters from two paraffin-section studies (PMC13450933; PMC3698551).
| Sample | FFPE human cancer tissue (datasheet A00520-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A00520-1); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-AKT3, 1:50-1:100 (datasheet A00520-1) |
| 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 | AKT3-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and nuclear expression at variable levels. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule; UniProt: nucleus). Neither article specifies retrieval conditions (PMC13450933; PMC3698551).
Section 2
What Is the Expected AKT3 Staining Pattern?
In paraffin-section IHC, expect variable cytoplasmic and nuclear AKT3 staining, including strong signal in selected neuronal, glial and glandular cells (HPA: tissue IHC). AKT3 has no transmembrane segment; UniProt also places it at membranes after stimulation (UniProt Q9Y243 topology and subcellular location). HPA rates its tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA: reliability).
| Strong cytoplasmic and nuclear staining in caudate or hippocampal neurons and cerebral cortex glia (HPA: High in these cells). | This fits the reported IHC pattern when the signal follows cell boundaries and recognizable nuclei (HPA: general cytoplasmic and nuclear expression; general IHC interpretation). Compare cells within the section: HPA reports variable levels, so uniform intensity across every cell is unnecessary (HPA: tissue IHC profile). |
| Signal appears only along cell borders, with no convincing cytoplasmic or nuclear staining. | Treat a membrane-only pattern cautiously: stimulated AKT3 can associate with membranes, but HPA describes general cytoplasmic and nuclear tissue staining (UniProt Q9Y243 subcellular location; HPA: tissue IHC profile). Check morphology and controls before calling it AKT3; the pattern alone cannot establish stimulation (general IHC interpretation). |
| Strong staining appears in salivary gland glandular cells or spleen red-pulp cells (HPA: Not detected in those cell populations). | This conflicts with the reported cell-level IHC observations and warrants checks for cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). It does not establish that every cell in either tissue must be negative (HPA: named cell populations). |
| Diffuse color covers stroma, blank areas and multiple cell types without clear cell boundaries. | This is difficult to score as AKT3 because HPA reports staining in identifiable cytoplasmic and nuclear compartments (HPA: tissue IHC profile). Review blocking, washing and detection controls for background; these are general chromogenic IHC checks, not documented AKT3-specific effects (general IHC practice). |
| No signal appears in caudate neurons or cerebral cortex glia despite preserved tissue morphology (HPA: High in those cells). | A negative result there needs technical review before biological interpretation (HPA: tissue IHC; general IHC practice). Verify antibody suitability for IHC-P, run a known-positive section, and review retrieval and detection conditions; the supplied sources do not identify an AKT3-specific retrieval requirement (general IHC practice). |
💡Expected AKT3 appearanceCall a convincing positive when identifiable caudate or hippocampal neurons, cerebral cortex glia, or reported glandular cells show cytoplasmic and nuclear staining that may be strong; widespread structureless color or strong signal in HPA-negative cell populations is suspect (HPA: tissue IHC; general IHC interpretation).
| Cell population and tissue context | HPA reports High staining in caudate and hippocampal neurons, cerebral cortex glia, and gallbladder and thyroid glandular cells; liver hepatocytes and heart cardiomyocytes are Low (HPA: tissue IHC). Select controls by the named cell population, since a whole-section label can hide cellular differences (general IHC interpretation). |
| Compartment and stimulation | UniProt lists nucleus, cytoplasm and membrane, with membrane association after stimulation; HPA's tissue IHC summary emphasizes cytoplasmic and nuclear staining (UniProt Q9Y243 subcellular location; HPA: tissue IHC profile). Membrane enrichment alone therefore needs context and cannot serve as a universal positive criterion. |
| Antibody evidence | HPA026441 and CAB013090 are both IHC Approved, while overall tissue staining has medium consistency with RNA expression (HPA: antibody validation and reliability). An Approved label supports use of the reported pattern, but does not make every unexpected stain specific (general IHC interpretation). |
| Isoforms and modified residues | UniProt lists 2 AKT3 isoforms and phosphorylation and glycosylation sites (UniProt Q9Y243 isoforms and PTMs). The payload gives no antibody epitope or isoform coverage, so these features cannot explain a particular positive or negative IHC result without antibody-specific evidence. |
| IF/ICC appearance | Where should IF/ICC signal appear? HPA mainly reports nucleoplasm and cytosol (supported) and Golgi apparatus (approved); plasma membrane, primary cilium and basal body are uncertain (HPA: subcellular ICC-IF). Those ICC-IF observations do not establish a required Golgi or ciliary pattern in paraffin-section IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive neuronal or glial cells show no chromogenic signal (HPA: High in specified brain cells). | Antibody, retrieval or detection conditions may have failed (general IHC practice); the sources give no AKT3-specific fixation sensitivity. | Check the catalog antibody's IHC-P instructions, known-positive section and detection controls; assess tissue preservation before interpreting absence as biological (general IHC practice). |
| Most of the section has a weak, even haze. | Nonspecific antibody binding or incomplete washing can raise background (general IHC practice). | Review blocking, antibody concentration and wash conditions, then compare with an appropriate primary-antibody omission control (general IHC practice). |
| Strong color appears in salivary gland glandular cells or spleen red-pulp cells (HPA: Not detected). | Cross-reactivity or endogenous detection activity is possible; HPA's cell-level negatives are reference observations, not proof of the mechanism (HPA: tissue IHC; general IHC practice). | Check detection-only controls and cell identity, then compare the result with a reported positive population (general IHC practice; HPA: tissue IHC). |
| Only sharp membrane outlines stain. | Membrane association can follow stimulation, but a membrane-only tissue pattern is not HPA's general IHC profile (UniProt Q9Y243 subcellular location; HPA: tissue IHC). | Inspect cytoplasmic and nuclear compartments, morphology and controls; avoid inferring activation from localization alone (general IHC interpretation). |
| Hepatocytes or cardiomyocytes stain faintly while a brain control stains strongly. | This can match HPA's Low staining in those cells and High staining in selected brain cells (HPA: tissue IHC). | Score the named cell populations separately and retain the positive control when comparing runs (HPA: tissue IHC; general IHC practice). |
| IF/ICC shows a Golgi-like signal that is inconspicuous in paraffin-section IHC. | HPA reports Golgi localization from ICC-IF, while its tissue IHC summary is cytoplasmic and nuclear (HPA: subcellular ICC-IF and tissue IHC). | Interpret each application against its own reported pattern; do not require Golgi staining to call an IHC section positive (HPA: ICC-IF and tissue IHC). |
Experiment evidence
IHC & IF images showing AKT3 staining
A00520-1 · IHCFormalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117020-AKT3/tissue/cerebral+cortex#imid_6676464
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117020-AKT3/tissue/colon#imid_6676190
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117020-AKT3/tissue/liver#imid_6676175
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117020-AKT3/tissue/kidney#imid_6676176
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB013090; green = AKT3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB013090; green = AKT3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AKT3 IHC & IF
🧪Run caudate first and score neuronal cells, which show High AKT3 staining (HPA: High in caudate neuronal cells). Use salivary gland glandular cells as the tissue negative (HPA: Not detected); on the caudate slide, assess non-neuronal cells without specific chromogen deposition as an internal background reference, without assuming every non-neuronal cell is AKT3-negative (HPA: neuronal cells scored High).
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Salivary gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AKT3 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Nucleoplasm (supported), Golgi apparatus (approved), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a host- and isotype-matched irrelevant IgG for a monoclonal antibody or matched nonimmune IgG for a polyclonal antibody; and AKT3-knockout material as a biological specificity control (standard IHC practice). Quench endogenous peroxidase for chromogenic detection; if examining caudate by IF, check tissue autofluorescence with an unstained control (standard IHC/IF practice).
⚠️Feasibility: The selected A00520-1 caption explicitly reports formalin-fixed, paraffin-embedded tissue and peroxidase/AEC detection, but reports no fixation duration or target-specific fixation window (selected-SKU tissue-IHC caption). AKT3 retrieval dependency and a target-specific fixation effect are unreported in the supplied evidence; optimize antigen retrieval empirically, and treat frozen-section performance as unreported (supplied target/application evidence). ICC/IF is documented in the listed HPA cell lines, while brain lipofuscin can contribute autofluorescent background when examining caudate by IF (HPA: ICC-IF image cell lines; standard IF practice).
HPA tissue IHC evidence for AKT3
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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Ciliated cells (ciliary rootlets) | High | Protein (IHC) | HPA → |
Section 3
Advanced AKT3 IHC Tips
Troubleshoot AKT3 staining in paraffin sections by checking retrieval, compartment pattern, cell identity, controls and scoring before interpreting differences between samples.
What retrieval should I start with for AKT3 in paraffin sections?
Start with Tris-EDTA at pH 9.0 for heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). Allow sections to cool consistently, then compare stained and no-primary controls processed in the same run (standard IHC practice). If staining is weak, test an alternative retrieval condition on matched sections while holding antibody concentration and detection constant (standard IHC practice). Score nuclear and cytoplasmic staining separately because both are reported for AKT3 in tissue (HPA: general cytoplasmic and nuclear expression). Excessively harsh retrieval can damage morphology, making compartment assignment unreliable (standard IHC practice).
Could fixation explain weak or uneven AKT3 staining?
The selected antibody has been shown on formalin-fixed, paraffin-embedded human cancer tissue (A00520-1 tissue-IHC caption). Its target-specific sensitivity to fixation duration or delay is unknown from the supplied evidence, so do not assign weak staining to those factors without a comparison. For troubleshooting, process matched sections with the same retrieval, antibody incubation and chromogenic detection, and record fixation history where available (standard IHC practice). Compare morphology alongside staining: damaged or poorly preserved regions are unreliable for scoring (standard IHC practice). Do not infer fixation tolerance from AKT3 phosphorylation sites or its reported cellular compartments (UniProt Q9Y243: modified residues and subcellular locations).
Which staining compartments are plausible for AKT3?
Evaluate nuclear and cytoplasmic staining as plausible tissue patterns (HPA: general cytoplasmic and nuclear expression at variable levels). AKT3 is also reported at membranes after stimulation, without a transmembrane segment (UniProt Q9Y243: subcellular location and topology). A membrane-enriched pattern therefore needs biological context and a control before it is interpreted as a change in AKT3 distribution (UniProt Q9Y243: stimulation-associated translocation; standard IHC practice). Record the compartment and cell type for each scored region rather than combining all chromogen into one intensity estimate (standard IHC practice). Isolated staining outside recognizable cells warrants inspection for precipitate or tissue damage (standard IHC practice).
Can this staining distinguish AKT3 isoforms or phosphorylation states?
AKT3 has 2 reported isoforms and PH, kinase and AGC-kinase C-terminal domains (UniProt Q9Y243: isoforms and domains). The supplied antibody evidence does not identify its epitope, so tissue staining alone cannot establish which isoform or domain is detected. AKT3 also has reported phosphorylation sites, including Thr305 and Ser472 (UniProt Q9Y243: modified residues). Treat chromogenic staining as AKT3 immunoreactivity unless antibody documentation demonstrates isoform or modification specificity (standard IHC interpretation). If an isoform-specific conclusion matters, check epitope coverage against the isoform sequences and use an independently validated control before assigning the signal (standard IHC practice).
How should I check an AKT3 tissue pattern with multiplex IF?
Use IF on matched tissue as a complementary check of the cell and compartment pattern observed by chromogenic IHC (standard IHC/IF practice). Pair AKT3 with a marker for the expected cell population, such as neuronal cells when examining caudate, where neuronal AKT3 staining is high (HPA: caudate neuronal cells). Choose a fluorophore separated from tissue autofluorescence, preferably after viewing an unstained section in each imaging channel (standard IF practice). Because the antibody epitope is unreported, test permeabilisation against a matched control when assessing intracellular signal; AKT3 has no transmembrane segment (UniProt Q9Y243: topology). Score concordance by cell and compartment, accounting for the different detection methods (standard IHC/IF practice).
How can I separate AKT3 staining from chromogenic background?
Run a no-primary section through the same peroxidase detection and chromogen steps to reveal detection background (standard IHC practice). The selected tissue-IHC image used a peroxidase-conjugated secondary antibody and AEC staining (A00520-1 tissue-IHC caption). A peroxidase block, appropriate protein block and controlled washing are general IHC measures; their effectiveness for this antibody is not established by that image (standard IHC practice). Compare background in blank tissue areas with signal inside intact cells, and review the counterstain before calling weak cytoplasmic color positive (standard IHC practice). If background persists, adjust one detection or blocking condition at a time on matched sections (standard IHC practice).
How should I score AKT3 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then record nuclear and cytoplasmic compartments separately (standard IHC practice; HPA: cytoplasmic and nuclear expression). An H-score can combine the percentage of cells at each intensity, while percentage positive is useful when intensity grading is unreliable (standard IHC practice). For sparse positive cells, report positive-cell density per mm² of evaluable tissue rather than per whole image (standard IHC practice). Normalize comparisons to the same cell population, tissue area, acquisition conditions and staining batch, with a reference section where feasible (standard IHC practice). Exclude folds, necrosis and damaged edges using the same rule for every specimen (standard IHC practice).
What makes an AKT3-positive result credible?
A credible result follows recognizable cellular boundaries and a plausible nuclear or cytoplasmic pattern (HPA: general cytoplasmic and nuclear AKT3 expression; standard IHC practice). Check the called cell type against tissue context: high staining is reported in caudate neuronal cells and cerebral cortex glial cells, while salivary glandular cells were not detected (HPA: tissue IHC). Be cautious with isolated rim staining, necrotic regions, edge effects or signal reproduced in the no-primary control, which can reflect artefact (standard IHC practice). Endogenous peroxidase must be considered in chromogenic detection, especially when no-primary controls stain (standard IHC practice). The selected cancer-tissue image demonstrates IHC use, but its clinical relevance was not evaluated (A00520-1 tissue-IHC caption).
Boster reagents
Best AKT3 / RAC-gamma serine/threonine-protein kinase IHC Antibodies
For AKT3, the catalog provides an IHC-P antibody with a human tissue image (catalog image caption) and a separate IF/ICC antibody listed for human samples (catalog applications/reactivity).
Real IHC data
Anti-AKT3 Antibody (Center)
Cat # A00520-1
A00520-1 is listed for IHC-P in human, monkey, mouse and rat, with an image of formalin-fixed, paraffin-embedded human cancer tissue (catalog applications/reactivity; catalog image caption). A00520-2 is listed for human IF/ICC, but has no IF image or reported fixative in the supplied payload (catalog applications/reactivity; catalog IF image alts).
Which to pick: Choose A00520-1 for chromogenic tissue IHC: its human tissue image shows peroxidase/AEC detection after formalin fixation and paraffin embedding, and its listed IHC-P dilution is 1:50–1:100 (catalog image caption; catalog IHC dilution). Choose A00520-2 for IF/ICC at the listed 5 μg/ml; its fixation conditions are unreported (catalog applications; catalog IF dilution; catalog IF image alts). For work across species, A00520-1 lists human, monkey, mouse and rat reactivity, while its supplied IHC image documents human tissue only (catalog reactivity; catalog image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y243 (AKT3_HUMAN, RAC-gamma serine/threonine-protein kinase).
- Human Protein Atlas. AKT3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. AKT3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm, the Golgi apparatus and cytosol. In addition localized to the plasma membrane, primary cilium and basal body..
- Human Protein Atlas. AKT3 antibody validation summary (2 antibodies).
- AKT3 and α-SMA in oral submucous fibrosis and oral squamous cell carcinoma with concomitant oral submucous fibrosis as potential prognostic indicators. Journal of oral and maxillofacial pathology : JOMFP 2026 — PMC13450933.
- Multifunctional Phase-Transition Nanoparticles for Effective Targeted Sonodynamic-Gene Therapy Against Thyroid Papillary Carcinoma. International journal of nanomedicine 2023 — PMC10163883.
- AKT3 drives adenoid cystic carcinoma development in salivary glands. Cancer medicine 2018 — PMC5806106.
- miR-29 targets Akt3 to reduce proliferation and facilitate differentiation of myoblasts in skeletal muscle development. Cell death & disease 2013 — PMC3698551.
- PubMed PMID:10092583 — UniProt-cited evidence.
- PubMed PMID:10208883 — UniProt-cited evidence.
- PubMed PMID:10491192 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AKT3 (RAC-gamma serine/threonine-protein kinase). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A00520-1 at 1:50-1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Salivary gland. IHC-P workflow on FFPE sections. Protocol steps: 1. Sample: FFPE human cancer tissue (datasheet A00520-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A00520-1); 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 Tris-EDTA buffer, pH 9.0 , 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-AKT3, 1:50-1:100 (datasheet A00520-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Use consistently formalin-fixed, paraffin-embedded sections (datasheet: A00520-1 IHC-P) (selected-SKU IHC image A00520-1); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; verify epitope coverage (UniProt); Regulation: Staining intensity varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt Q9Y243, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm, the Golgi apparatus and…, Datasheet A00520-1, PMC13450933, PMC3698551. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AKT3/immunohistochemistry-ihc-guide.