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AKT1 / RAC-alpha serine/threonine-protein kinase · IHC design guide
Design Immunohistochemistry for AKT1
Use the ubiquitous nuclear tissue profile as a benchmark for AKT1 paraffin-section IHC (HPA tissue IHC). This guide covers fixation, detection and scoring with an IHC-validated total-AKT1 antibody (datasheet: M00024-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AKT1 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 | Ubiquitous nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent and avoid overfixation (standard IHC practice; not target-specific) | |
| Caveat | Activation can redistribute AKT1 to nuclei (UniProt) | |
| Regulation | Higher in malignant prostate (UniProt) | |
| Isoform / epitope | Two isoforms; antibody epitope coverage is unspecified (UniProt; datasheet: M00024-1) |
Section 1
Recommended AKT1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with 2 published AKT1 IHC protocols (PMC3369856; PMC9600081).
| Sample | Paraffin-embedded human colon tissue; fixative not specified (datasheet M00024-1) |
| Fixation | Image fixative and duration unreported (datasheet M00024-1); 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 monoclonal (clone EBG-1) anti-AKT1, 1:50 (datasheet M00024-1) |
| 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 | AKT1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval); PMC9600081 reports a different retrieval procedure.
Section 2
What Is the Expected AKT1 Staining Pattern?
AKT1 has a predominantly nuclear tissue IHC pattern (HPA: Supported; high consistency between staining and RNA data). Expect staining in many cell types, including adrenal and breast glandular cells, bronchial respiratory epithelial cells, and cerebral cortical neurons (HPA: High in each). Cytoplasmic and activation-dependent membrane localization are also biologically plausible (UniProt P31749: subcellular location). AKT1 has no transmembrane segment (UniProt P31749: topology).
| Nuclear staining across several tissue compartments, including clearly stained glandular cells. | This fits the ubiquitous nuclear tissue profile (HPA: Supported). High staining in adrenal and breast glandular cells provides useful tissue context (HPA: High). Judge the signal in identifiable cells against the counterstain and local background; a brown deposit without a clear cellular boundary is not sufficient for a localization call (standard IHC practice). |
| Cytoplasmic staining accompanies nuclear staining; some cells show membrane-associated signal. | Cytoplasmic localization is recorded for AKT1, and activated AKT1 can move to the cell membrane and subsequently the nucleus (UniProt P31749: subcellular location). A total-AKT1 IHC pattern alone cannot establish phosphorylation or activation state (UniProt P31749: activation-linked localization; standard IHC interpretation). Review membrane-like staining in its cellular context before scoring it as specific. |
| Predominantly extracellular staining, or pigment over tissue-free areas, is called a positive result. | That distribution does not match AKT1's recorded cellular locations or lack of a transmembrane segment (UniProt P31749: subcellular location and topology). Check whether deposit follows tissue edges, damaged areas, or reagent pooling (standard IHC practice). Do not discard all nonnuclear signal automatically: cytoplasmic and condition-dependent membrane localization are plausible (UniProt P31749). |
| Strong staining appears in an unexpected cell population while recognizable cells show a different pattern. | AKT1 is reported in all human cell types analyzed, so an unexpected stained cell type alone does not prove cross-reactivity (UniProt P31749: tissue specificity). Compare its compartment and intensity with nearby cells and an independent control (standard IHC practice). Endogenous detection activity or antibody cross-reactivity becomes more concerning when the signal is diffuse, misplaced, or persists in a reagent control (standard IHC practice). |
| No convincing signal appears in an intact, known-positive section. | High staining is reported in adrenal glandular cells, appendix lymphoid tissue, bone-marrow hematopoietic cells, and other listed populations (HPA: High). First confirm that the expected cells are present and that the detection run worked (standard IHC practice). A negative result under one staining condition does not override the reported tissue pattern (HPA: Supported; standard IHC interpretation). |
💡Expected AKT1 appearanceCall a positive result when identifiable cells show nuclear staining above local background, with strong signal plausible in HPA High populations; diffuse extracellular deposit or tissue-free pigment is a false-positive pattern (HPA: ubiquitous nuclear expression; HPA: High; UniProt P31749: cellular locations; standard IHC practice).
| Tissue and cell selection | Use an identifiable HPA High population to judge a run, such as adrenal glandular cells or cerebral cortical neurons (HPA: High). Salivary glandular cells and skeletal myocytes are reported Low and are weaker choices for ruling out a failed stain (HPA: Low). The HPA tissue profile is Supported, meaning staining and RNA data show high consistency (HPA: reliability description). |
| Cellular compartment and activation | The tissue IHC profile emphasizes nuclei, while UniProt also lists cytoplasm and cell membrane (HPA: ubiquitous nuclear expression; UniProt P31749: subcellular location). UniProt links membrane localization and later nuclear translocation to activation-related phosphorylation (UniProt P31749). Neither compartment alone establishes the activation state of a section stained for total AKT1 (standard IHC interpretation). |
| Antibody validation and target identity | The listed antibodies HPA002891, CAB003765, and CAB080436 each have Supported IHC status (HPA: antibody validation). AKT1 is one of three closely related AKT kinases, and two AKT1 isoforms are recorded (UniProt P31749: function and isoforms). Confirm the particular antibody's documented specificity before assigning an unusual pattern uniquely to AKT1 (standard IHC practice). |
| What should IF/ICC show? | HPA reports supported nucleoplasm localization in ICC-IF; it also lists other locations with varying confidence, including uncertain microtubules and approved principal and end pieces (HPA: subcellular summary). IF/ICC images and validation do not replace the tissue IHC pattern or establish an IHC-P protocol (HPA: subcellular and tissue profiles). Interpret IF/ICC on its separate guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected nuclei are pale in a high-staining tissue. | The selected area may lack the relevant cells, or the IHC run may have inadequate signal (HPA: High in listed cell populations; standard IHC practice). | Locate the specified cell population on the counterstained section, then check a known-positive control and the run's detection steps before interpreting the sample (HPA: High; standard IHC practice). |
| Brown signal spreads evenly across stroma and empty spaces. | Diffuse deposit is inconsistent with the reported intracellular locations of AKT1 (UniProt P31749: subcellular location); nonspecific reagent background is possible (standard IHC practice). | Compare with a reagent control, inspect tissue edges and pooling, and review blocking, washes, and chromogen development (standard IHC practice). Score only signal that resolves to cells and compartments. |
| A strong cytoplasmic or membrane pattern seems to conflict with the nuclear reference. | Both compartments are listed for AKT1, while the HPA tissue profile summarizes a ubiquitous nuclear pattern (UniProt P31749: subcellular location; HPA: tissue profile). | Record nuclear and nonnuclear compartments separately. Compare with the validated IHC pattern and local controls; avoid inferring phosphorylation from total-AKT1 staining (HPA: Supported IHC; standard IHC interpretation). |
| Signal appears chiefly outside cells or along damaged section edges. | AKT1 has no transmembrane segment and is assigned cellular locations, making extracellular-only staining implausible (UniProt P31749: topology and subcellular location). Edge deposit can reflect a staining artifact (standard IHC practice). | Inspect intact fields and a reagent control, then review section handling, washing, and chromogen development (standard IHC practice). Exclude damaged edges from scoring. |
| A low-staining tissue is negative while the run control is positive. | Salivary glandular cells and skeletal myocytes are reported Low, so absence of an obvious signal there is less informative than loss in a High population (HPA: Low; HPA: High). | Judge assay performance in an identifiable HPA High population on a suitable control section; report the low-staining tissue result with its cell context (HPA: tissue IHC; standard IHC practice). |
| Unexpected cells stain despite a clean background. | Broad AKT1 expression is reported, so cell identity by itself does not establish an error (UniProt P31749: tissue specificity). Antibody cross-reactivity remains a possibility for an unsupported pattern (standard IHC practice). | Check compartment, morphology, and the antibody's IHC validation; compare with a suitable independent control before attributing the signal specifically to AKT1 (HPA: Supported IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing AKT1 staining
M00024-1 · IHCImmunohistochemical analysis of paraffin-embedded human colon, using AKT1 Antibody.
M00024-1 · IFImmunofluorescent analysis of Hela cells, using AKT1 Antibody.
P00024-2 · IHCIHC analysis of AKT1 using anti-AKT1 antibody (P00024-2). AKT1 was detected in a paraffin-embedded section of human colorectal adenocarcinom
P00024-2 · IHCIHC analysis of AKT1 using anti-AKT1 antibody (P00024-2). AKT1 was detected in a paraffin-embedded section of human prostate cancer tissue.
P00024-2 · IFIF analysis of AKT1 using anti-AKT1 antibody (P00024-2) and anti-Beta Tubulin antibody (M01857-3). AKT1 was detected in immunocytochemical s
P00024-5 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using Phospho-Akt(Ser473) Antibody.
P00024-5 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000142208-AKT1/tissue/cerebral+cortex#imid_945056
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000142208-AKT1/tissue/colon#imid_944974
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000142208-AKT1/tissue/liver#imid_944983
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000142208-AKT1/tissue/kidney#imid_944992
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002891; green = AKT1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002891; green = AKT1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AKT1 IHC & IF
🧪Run breast first and assess its glandular cells for AKT1 staining (HPA: High in breast glandular cells). HPA detects AKT1 in all 45 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and expect any unstained cells within the breast section to show counterstain only without designating them AKT1-negative (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: AKT1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AKT1 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Nucleoplasm (supported), Microtubules (uncertain), Principal piece (approved), End piece (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a control matched to the primary antibody’s host species and isotype, or species-matched nonimmune IgG for a polyclonal primary; a matched AKT1 knockout sample can test biological specificity (standard IHC practice). For chromogenic breast IHC, quench endogenous peroxidase and use an appropriate control if a biotin-based detection system is used; for breast IF, assess tissue autofluorescence with a no-primary slide (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative for the M00024-1 paraffin-section caption is unreported (selected-SKU caption). Antigen retrieval may need empirical optimization for paraffin IHC; the supplied evidence does not establish a retrieval dependency or show that frozen sections or IF are easier (selected-SKU caption; standard IHC practice). Breast tissue autofluorescence may complicate IF interpretation, so check the no-primary slide (standard IF practice).
HPA tissue IHC evidence for AKT1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: AKT1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced AKT1 IHC Tips
Troubleshoot AKT1 staining by checking retrieval, compartment patterns, antibody specificity, and controls before comparing chromogenic IHC results across sections.
What should I change when AKT1 staining is weak after antigen retrieval?
Start with Tris-EDTA pH 9.0 heat retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Let sections cool in retrieval buffer before washing, and keep heating and cooling conditions consistent across the comparison set (standard IHC practice). The selected AKT1 image documents paraffin-embedded human colon but does not report its fixative or retrieval method, so it cannot establish a product-specific optimum (caption: M00024-1). If staining remains weak, compare a shorter or longer retrieval time on adjacent sections while holding antibody dilution and detection constant (standard IHC practice). Check tissue integrity alongside signal because excessive heating can damage morphology and complicate nuclear scoring (standard IHC practice).
Could fixation explain weak or uneven AKT1 staining in paraffin sections?
Target-specific AKT1 sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-embedded human colon caption does not state a fixative (caption: M00024-1). Record the actual fixative, fixation duration, processing history, and section age for each specimen before attributing a difference to AKT1 biology (standard IHC practice). Compare adjacent sections processed together, using the same Tris-EDTA pH 9.0 retrieval and detection conditions to isolate specimen effects (page retrieval rule; standard IHC practice). Examine morphology for uneven preservation and compare staining in intact regions rather than damaged edges (standard IHC practice). Do not infer fixation sensitivity from AKT1 localisation or modification sites, which provide no direct fixation experiment (UniProt P31749 record).
Should AKT1 appear in nuclei, cytoplasm, or at the cell membrane?
Expect nuclear staining to be plausible in tissue IHC: the tissue profile reports ubiquitous nuclear expression, while the subcellular profile supports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Cytoplasmic and cell-membrane localisation are also reported for AKT1, and nuclear localisation can follow activation (UniProt P31749 localisation). Score nuclear, cytoplasmic, and membrane-associated staining separately instead of combining them into one intensity value (standard IHC practice; UniProt P31749 localisation). Compare compartments within morphologically intact cells and include a no-primary control when a new compartment pattern appears (standard IHC practice). A total-AKT1 stain alone does not establish phosphorylation status or pathway activation; that interpretation requires a separately validated phospho-specific readout (UniProt P31749 activation description; standard IHC practice).
Can this stain distinguish AKT1 isoforms or phosphorylation states?
AKT1 has 2 listed isoforms, but the supplied caption gives no immunogen, epitope, or isoform-reactivity data for M00024-1 (UniProt P31749 isoforms; caption: M00024-1). Its PH domain spans residues 5–108, kinase domain 150–408, and C-terminal domain 409–480, so epitope position matters when assessing coverage (UniProt P31749 domains). AKT1 also carries reported modification sites, including phosphorylation relevant to activation at Thr-308 and Ser-473 (UniProt P31749 record). Do not label chromogenic intensity as isoform-specific or phospho-specific without antibody documentation and appropriate controls (standard IHC practice). If that distinction drives the experiment, compare independently validated reagents on matched sections and report each reagent’s defined target (standard IHC practice).
How should I check the AKT1 pattern with multiplex immunofluorescence?
Use IF on a matched section to inspect AKT1 compartments while retaining chromogenic IHC as this page’s primary readout (standard IHC/IF practice). Pair AKT1 with a validated marker for the expected cell population, such as a glandular-cell marker when examining breast glandular cells, which show high tissue staining (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a far-red channel after checking tissue autofluorescence and single-colour controls (standard IF practice). AKT1 has no transmembrane segment and occurs in nucleus, cytoplasm, and at the cell membrane; use mild permeabilisation for intracellular access while checking for pattern changes (UniProt P31749 topology/localisation; standard IF practice). Because the antibody epitope is unspecified, verify its IF performance independently before interpreting colocalisation (caption: M00024-1; standard IF practice).
How can I separate broad AKT1 expression from chromogenic background?
Broad staining is plausible because the tissue profile describes ubiquitous nuclear AKT1 expression and low tissue specificity at the RNA level (HPA tissue IHC). Include a no-primary section to reveal detection-system staining, and apply an endogenous-peroxidase block before HRP and DAB detection (standard chromogenic IHC practice). Use matched processing and DAB development times so diffuse colour does not reflect a longer reaction in one section (standard IHC practice). Salivary-gland glandular cells and skeletal-muscle myocytes are reported as low, so they may serve as relative comparisons but not guaranteed negatives (HPA tissue IHC). If background persists, optimise blocking, antibody concentration, washes, and detection exposure one variable at a time (standard IHC practice).
What is a defensible way to quantify AKT1 IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because AKT1 may be nuclear, cytoplasmic, or membrane-associated (UniProt P31749 localisation; HPA tissue IHC). For nuclear staining, report the percentage of positive viable target cells and an H-score using intensity grades 0–3, yielding a 0–300 scale (standard IHC practice). For a spatial question, report positive-cell density per mm² of viable, annotated tissue rather than raw counts per image (standard IHC practice). Normalise comparisons to the same target-cell population or viable tissue area, and use identical thresholds, counterstain conditions, and development time (standard IHC practice). Keep compartment scores separate and document excluded necrotic or damaged regions before interpreting group differences (standard IHC practice).
When is an apparent AKT1-positive area more likely to be artefact?
Treat nuclear staining in intact cells as biologically plausible because the tissue profile reports ubiquitous nuclear expression and the subcellular profile supports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Cytoplasmic or membrane-associated staining can also fit AKT1 localisation, but a total-AKT1 pattern does not by itself establish activation (UniProt P31749 localisation/activation; standard IHC practice). Check that positive cells have interpretable morphology and compare their distribution with the expected cell population on the counterstained section (standard IHC practice). Strong colour confined to section edges, folds, or necrotic tissue should trigger review of section quality and staining uniformity (standard IHC practice). Colour persisting in a no-primary control suggests background, including endogenous enzyme activity, rather than antibody-dependent AKT1 detection (standard chromogenic IHC practice).
Boster reagents
Best AKT1 / RAC-alpha serine/threonine-protein kinase IHC Antibodies
Three catalog antibodies have IHC images from human paraffin sections and IF images from cultured cells (catalog image captions); all list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-AKT1 Monoclonal Antibody
Cat # M00024-1
Real IHC data
Anti-Phospho-AKT1 (T450) Rabbit Monoclonal Antibody
Cat # P00024-2
Real IHC data
Anti-Phospho-Akt (Ser473) AKT1 Rabbit Monoclonal Antibody
Cat # P00024-5
M00024-1 has IHC data from human colon and IF data from HeLa cells (M00024-1 image captions). P00024-2 has IHC data from human colorectal adenocarcinoma and IF data from A549 cells; P00024-5 has IHC data from human kidney and IF data from NIH/3T3 cells (respective image captions).
Which to pick: For paraffin-section IHC, choose M00024-1 for total AKT1, P00024-2 for phospho-T450, or P00024-5 for phospho-Ser473 (catalog titles and each SKU’s IHC caption); the captions do not report the fixative. For IF/ICC, all three list those applications, with IF images from HeLa, A549 and NIH/3T3 cells, respectively (catalog applications and IF captions). All three are rabbit monoclonals that list human, mouse and rat reactivity, but their IHC captions document human tissue only (catalog host, clone, reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P31749 (AKT1_HUMAN, RAC-alpha serine/threonine-protein kinase).
- Human Protein Atlas. AKT1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. AKT1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm, microtubules, principal piece and end piece. In addition localized to the primary cilium, basal body, perinuclear theca and calyx..
- Human Protein Atlas. AKT1 antibody validation summary (3 antibodies).
- Network pharmacology and bioinformatics analysis-based strategy for exploring the mechanisms of luteolin in the treatment of five types of cancer: A retrospective computational study. The Journal of international medical research 2026 — PMC13309665.
- Phosphorylated Akt1 expression is associated with poor prognosis in cutaneous, oral and sinonasal melanomas. Oncotarget 2018 — PMC6324666.
- AKT1 loss correlates with episomal HPV16 in vulval intraepithelial neoplasia. PloS one 2012 — PMC3369856.
- Expression of AKT1 Related with Clinicopathological Parameters in Clear Cell Renal Cell Carcinoma. Current issues in molecular biology 2022 — PMC9600081.
- PubMed PMID:1851997 — UniProt-cited evidence.
- PubMed PMID:11508278 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AKT1 (RAC-alpha serine/threonine-protein kinase). Expected localisation Ubiquitous 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: M00024-1 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon tissue; fixative not specified (datasheet M00024-1); 2. Fixation: Image fixative and duration unreported (datasheet M00024-1); 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 monoclonal (clone EBG-1) anti-AKT1, 1:50 (datasheet M00024-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent and avoid overfixation (standard IHC practice; not target-specific); Caveat: Activation can redistribute AKT1 to nuclei (UniProt); Isoform / epitope: Two isoforms; antibody epitope coverage is unspecified (UniProt; datasheet: M00024-1); Regulation: Higher in malignant prostate (UniProt). Evidence: UniProt P31749, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm, microtubules, principal piece…, Datasheet M00024-1, PMC3369856, PMC9600081. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AKT1/immunohistochemistry-ihc-guide.