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PLEK / Pleckstrin · IHC design guide
Design Immunohistochemistry for PLEK
Plan chromogenic PLEK IHC in paraffin sections with the IHC-validated antibody (datasheet A06656-3). This guide highlights marrow and lymph node staining and helps select tissue controls and score cell populations (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PLEK 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 extracellular in marrow and lymph node (HPA tissue IHC) | |
| Staining pattern | Marrow hematopoietic and nodal germinal center cells; cytoplasmic and extracellular staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06656-3) | |
| Positive control | Lymph node+4 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 | Myeloid peroxidase may raise marrow DAB background (standard IHC practice) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope |
Section 1
Recommended PLEK IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A06656-3) is followed by published PLEK IHC protocols for placenta, carotid plaque, and gingival tissue (PMC11979283; PMC10929796; PMC8787058).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A06656-3) |
| Fixation | Image fixative and duration unreported (datasheet A06656-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A06656-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06656-3) |
| Primary antibody | Rabbit anti-PLEK, 2-5 μg/ml (datasheet A06656-3) |
| Primary incubation | Overnight at 4 °C (datasheet A06656-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06656-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PLEK-positive staining in germinal center cells of lymph node (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and extracellular expression in bone marrow and lymph node. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet: A06656-3); citrate pH 6 is a published alternative for gingival sections (PMC8787058).
Section 2
What Is the Expected PLEK Staining Pattern?
In paraffin-section IHC, expect PLEK in the cytoplasm of selected hematopoietic cells, with extracellular staining also reported in bone marrow and lymph node (HPA tissue IHC). Germinal center cells in lymph node and red-pulp cells in spleen show high staining; bone-marrow hematopoietic cells show medium staining (HPA tissue IHC). HPA rates tissue-IHC reliability Enhanced, with medium RNA–staining consistency (HPA tissue IHC). PLEK has no transmembrane segment (UniProt P08567 topology).
| Strong cytoplasmic signal in lymph-node germinal center cells or splenic red-pulp cells. | This fits the high-staining cell populations reported for PLEK (HPA tissue IHC). Judge the labeled cells against adjacent tissue architecture: a positive field need not imply that every cell in the section expresses PLEK (HPA tissue IHC; standard IHC interpretation). |
| Extracellular staining in bone marrow or lymph node, alongside a plausible cellular pattern. | HPA includes extracellular expression in its tissue-IHC summary, so extracellular signal alone is not grounds for rejection (HPA tissue IHC). Assess its distribution and the cellular signal together; the summary does not identify an extracellular source or mechanism (HPA tissue IHC). |
| Predominantly nuclear or sharply membranous chromogen in the expected positive cells. | That differs from HPA's cytoplasmic and extracellular tissue-IHC summary and warrants a specificity check (HPA tissue IHC). Do not equate an IHC nuclear pattern with HPA's approved nucleolar ICC-IF result: the assays report different observations (HPA subcellular ICC-IF). |
| Broad staining of cell types listed as undetected, such as adipocytes or respiratory epithelial cells. | HPA reports no detection in adipocytes of adipose tissue or respiratory epithelial cells of bronchus (HPA tissue IHC). Review morphology and controls for cross-reactivity or endogenous detection activity before interpreting widespread staining as PLEK (standard IHC practice). |
| Little or no signal in a lymph-node germinal center or splenic red-pulp control. | These are reported high-staining populations, so an absent result weakens confidence in a negative test section (HPA tissue IHC). Check section quality and the IHC detection workflow before concluding that the tested tissue lacks PLEK (standard IHC practice). |
💡Expected PLEK appearanceA convincing positive IHC result shows strong cytoplasmic staining in lymph-node germinal center or splenic red-pulp cells, with reported extracellular signal assessed in context; indiscriminate staining of HPA-undetected cell types is suspect (HPA tissue IHC; standard IHC interpretation).
| Tissue and cell population | HPA reports high staining in lymph-node germinal center and splenic red-pulp cells, medium staining in bone-marrow hematopoietic cells, and no detection in adipose-tissue adipocytes (HPA tissue IHC). Choose controls by cell population, not tissue name alone (standard IHC practice). |
| Evidence strength | HPA rates both HPA031838 and HPA057341 Enhanced for IHC, while describing medium consistency between antibody staining and RNA expression (HPA antibodies; HPA tissue IHC). Treat the tissue pattern as a reference that still requires appropriate slide controls (standard IHC practice). |
| Topology and processing | UniProt lists one 1–350 pleckstrin chain, no signal peptide or propeptide, and no transmembrane segment (UniProt P08567). These features do not establish the source of HPA's extracellular IHC signal or a paraffin-section retrieval condition (UniProt P08567; HPA tissue IHC). |
| IF/ICC Q: Is nucleolar signal expected? | A: HPA approves nucleolar ICC-IF localization and lists HEL, NB4 and U2OS image sets; HPA057341 is ICC Approved (HPA subcellular ICC-IF; HPA antibodies). That finding does not replace the cytoplasmic and extracellular tissue-IHC reference pattern (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogen in the expected positive-control cells. | An IHC workflow failure is possible when a population reported as high staining is blank (HPA tissue IHC; standard IHC practice). | Confirm that the control contains recognizable germinal center or red-pulp cells, then check deparaffinization, the antibody's IHC-P retrieval instructions, detection reagents and counterstain (standard IHC practice). |
| Diffuse chromogen obscures cell boundaries. | Excess background can arise from inadequate blocking, excessive primary antibody or incomplete washing (standard IHC practice). HPA's extracellular summary alone cannot identify the cause (HPA tissue IHC). | Review a no-primary control, blocking and wash steps; adjust the primary-antibody condition within the validated IHC workflow, then reassess the cellular pattern (standard IHC practice). |
| Chromogen appears in many HPA-undetected cell types. | Cross-reactivity or endogenous detection activity may explain a pattern unlike HPA's listed negatives (HPA tissue IHC; standard IHC practice). | Compare the suspect section with a no-primary control and an appropriate positive tissue; check endogenous-enzyme blocking for the chosen chromogenic detection system (standard IHC practice). |
| Signal looks exclusively nuclear in a paraffin section. | Nuclear-only tissue IHC does not match HPA's cytoplasmic and extracellular summary, although HPA separately approves nucleolar ICC-IF localization (HPA tissue IHC; HPA subcellular ICC-IF). | Check nuclear morphology, counterstain and no-primary control; compare the same cell population with an appropriate positive tissue before assigning the signal to PLEK (standard IHC practice). |
| Only extracellular material stains in bone marrow or lymph node. | HPA reports extracellular expression but gives no mechanism or criterion that makes extracellular-only staining sufficient for a positive call (HPA tissue IHC). | Examine the reported hematopoietic or germinal center cell populations and a no-primary control; describe extracellular-only signal separately from cellular positivity (HPA tissue IHC; standard IHC interpretation). |
| A weak test section is difficult to score. | PLEK staining varies by cell population: HPA reports high, medium, low and undetected examples, and notes medium RNA–staining consistency (HPA tissue IHC). | Score the relevant cell type against a concurrently stained high-positive control and record intensity and distribution; avoid calling the whole tissue negative from a sparse field (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing PLEK staining
A06656-3 · IHCIHC analysis of Pleckstrin/PLEK using anti-Pleckstrin/PLEK antibody (A06656-3). Pleckstrin/PLEK was detected in a paraffin-embedded section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115956-PLEK/tissue/cerebral+cortex#imid_20849455
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115956-PLEK/tissue/colon#imid_20849173
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115956-PLEK/tissue/liver#imid_20849174
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000115956-PLEK/tissue/kidney#imid_20849175
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA057341; green = PLEK, blue = nucleus, red = microtubules.
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA057341; green = PLEK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PLEK IHC & IF
🧪Run lymph node first and look for staining in germinal center cells (HPA: High in germinal center cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the lymph node slide, cells without specific staining outside the germinal centers can serve as internal background references, but their PLEK status is not established by the supplied HPA row.
Positive control tissue: Lymph node (Germinal center cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PLEK in HEL, NB4, U2OS, with annotated localisation: Nucleoli (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species- and isotype-matched rabbit control immunoglobulin alongside a PLEK knockout specimen or cognate-peptide blocking control (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase before HRP–DAB detection, particularly when assessing background in lymph node tissue (caption: HRP–DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06656-3 paraffin-section caption does not state the fixative (caption: fixative not stated). The documented IHC procedure uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish that retrieval is required for every specimen (caption: EDTA heat retrieval). Frozen sections and IF are not established as easier by the supplied evidence; in lymph node, endogenous peroxidase can complicate interpretation of DAB background (caption: HRP–DAB detection; standard IHC practice).
HPA tissue IHC evidence for PLEK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Paneth cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PLEK IHC Tips
Troubleshoot PLEK staining in paraffin sections by checking retrieval, cell identity, compartment, controls, and scoring before interpreting signal.
Which retrieval conditions should I start with for PLEK in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin-section PLEK IHC (datasheet A06656-3). The selected tonsil image used those conditions, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (caption A06656-3). Run a tonsil control alongside the test section and compare germinal center staining, which HPA reports at a medium level (HPA: tonsil germinal center cells). If signal is weak, check retrieval temperature, heating time, section adhesion, and reagent performance before testing another buffer as a fallback (standard IHC practice). Keep retrieval conditions identical across sections intended for comparison (standard IHC practice).
How should I assess whether fixation is affecting PLEK staining?
Target-specific fixation sensitivity is unknown: the selected PLEK tissue image identifies a paraffin section but does not state its fixative (caption A06656-3). Record the actual fixative, fixation duration, processing history, and section age for each specimen before comparing chromogenic signal (standard IHC practice). When staining varies, process a known positive control with the test material and hold retrieval, antibody concentration, detection, and development conditions constant (standard IHC practice). Compare staining within the same cell population, such as tonsil germinal center cells, rather than across unlike compartments (HPA: medium in tonsil germinal center cells). Do not assign a PLEK-specific fixation effect without a controlled comparison (caption A06656-3; standard IHC practice).
What cellular pattern should I expect in PLEK chromogenic IHC?
Assess the stained cell type and compartment separately: HPA describes cytoplasmic and extracellular tissue expression in bone marrow and lymph node (HPA: tissue IHC profile). HPA also reports nucleolar localisation from ICC/IF images, while UniProt supplies no subcellular annotation for PLEK (HPA: subcellular; UniProt P08567). These observations do not establish that every paraffin-section signal should be nucleolar or extracellular (HPA: tissue IHC profile; HPA: subcellular). PLEK has no annotated transmembrane segment, so a crisp cell-surface outline alone warrants scrutiny (UniProt P08567 topology). Check morphology and a no-primary control before assigning unexpected nuclear, surface, or extracellular DAB deposits to PLEK (standard IHC practice).
Could epitope placement or PLEK modification explain discordant IHC staining?
Map the antibody’s stated immunogen, if available, against PLEK’s PH domains at residues 4–101 and 244–347, and DEP domain at 136–221 (UniProt P08567 domains). The supplied record lists 0 annotated isoforms; that does not prove every specimen presents an identical accessible epitope (UniProt P08567 isoforms; standard IHC practice). PLEK includes annotated acetylation at residue 64 and PKC-linked phosphorylation at 113 and 117, but their effect on this antibody’s IHC staining is unknown (UniProt P08567 modified residues). Compare serial-section staining after the established EDTA pH 8.0 retrieval before attributing a difference to modification (datasheet A06656-3; standard IHC practice). An independent antibody recognizing a documented different epitope can help test specificity (standard IHC practice).
How can IF help investigate an ambiguous PLEK IHC pattern?
Use IF as a separate localisation check when DAB cannot resolve whether staining belongs to a particular cell or compartment (standard IHC/IF practice). Multiplex PLEK with a validated marker for the cell population under study; tonsil germinal center cells are a reported IHC-positive population (HPA: medium in tonsil germinal center cells). Choose fluorophores after inspecting unstained tissue autofluorescence, and include single-label controls to assess spectral overlap (standard IF practice). Because PLEK has no annotated transmembrane segment, plan permeabilisation around the intracellular epitope and marker, then verify that treatment preserves morphology (UniProt P08567 topology; standard IF practice). HPA’s nucleolar ICC/IF observation is a useful comparison, not a substitute for validating the paraffin-section result (HPA: subcellular; standard IHC/IF practice).
What should I check when PLEK DAB staining looks diffuse or widespread?
First compare the stained slide with no-primary and detection-only controls to separate primary-antibody signal from reagent or tissue background (standard IHC practice). Check the peroxidase block and DAB development when deposits appear outside cells or across the section, since endogenous enzyme activity and excess development can contribute to chromogenic background (standard IHC practice). The selected tonsil image used 10% goat serum blocking, 2 μg/ml primary antibody, and DAB development, providing documented starting conditions for this SKU (caption A06656-3). If background remains high, assess blocking, washes, primary concentration, and development time one variable at a time (standard IHC practice). Compare suspected signal with morphology and expected positive cells before scoring it (HPA: tonsil germinal center cells; standard IHC practice).
How should I score PLEK staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the target cell population and compartment before scoring, because HPA reports different PLEK levels across cell types and tissues (HPA: tissue IHC profile). For a defined population, record the percentage of positive cells and an intensity-based H-score; use positive-cell density per mm² when spatial abundance is the question (standard IHC scoring practice). Normalize cell counts to the annotated tissue area or eligible cells, and exclude folds, necrosis, and section edges using the same rule across samples (standard IHC scoring practice). Keep retrieval, staining batch, DAB development, imaging, and positivity thresholds consistent, with a shared control section for batch comparison (standard IHC practice). Report cytoplasmic and nucleolar scores separately if both are evaluated (HPA: tissue IHC profile; HPA: subcellular).
How do I distinguish true PLEK staining from artefact?
A credible result combines reproducible cell-specific staining, suitable controls, and morphology consistent with the scored compartment (standard IHC practice). Tonsil germinal center cells show medium PLEK staining in HPA and appear in the selected paraffin-section image, making them a relevant comparison for this antibody (HPA: tonsil germinal center cells; caption A06656-3). Treat staining confined to section edges, damaged or necrotic areas, or no-primary controls as suspect; check the peroxidase block if DAB appears without primary antibody (standard IHC practice). Investigate an isolated membrane-outline pattern because PLEK has no annotated transmembrane segment, while recognising that HPA’s tissue and ICC/IF compartment reports differ (UniProt P08567 topology; HPA: tissue IHC profile; HPA: subcellular). Interpret a negative field in light of its specific cell population and the matched positive control (standard IHC practice).
Boster reagents
Best PLEK / Pleckstrin IHC Antibodies
A06656-3 has real chromogenic IHC data from a paraffin-embedded human tonsil section (A06656-3 IHC image caption); human is its only listed species (catalog reactivity).
Real IHC data
Anti-Pleckstrin/PLEK Antibody ®
Cat # A06656-3
A06656-3 was demonstrated on a paraffin-embedded human tonsil section using EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A06656-3 IHC image caption). IHC is listed as an application, while IF/ICC is not, and no IF image is supplied (catalog applications; catalog IF image alts).
Which to pick: Choose A06656-3 for human paraffin-section IHC; the catalog lists 2–5 μg/ml, and its own tonsil image used 2 μg/ml (catalog IHC dilution; A06656-3 IHC image caption). The caption does not report the fixative, and the catalog provides no IF/ICC validation for this SKU (A06656-3 IHC image caption; catalog applications and IF image alts). No cross-species choice is supported: A06656-3 lists human reactivity only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P08567 (PLEK_HUMAN, Pleckstrin).
- Human Protein Atlas. PLEK tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. PLEK subcellular location (ICC-IF): Localized to the nucleoli..
- Human Protein Atlas. PLEK antibody validation summary (2 antibodies).
- Using weighted gene co-expression network analysis to identify key genes related to preeclampsia. Frontiers in immunology 2025 — PMC11979283.
- Comprehensive analysis identifies crucial genes associated with immune cells mediating progression of carotid atherosclerotic plaque. Aging 2024 — PMC10929796.
- Pleckstrin Levels Are Increased in Patients with Chronic Periodontitis and Regulated via the MAP Kinase-p38α Signaling Pathway in Gingival Fibroblasts. Frontiers in immunology 2021 — PMC8787058.
- Integrating machine learning algorithms and single-cell analysis to identify gut microbiota-related macrophage biomarkers in atherosclerotic plaques. Frontiers in cellular and infection microbiology 2024 — PMC11074432.
- PubMed PMID:2897630 — UniProt-cited evidence.
- PubMed PMID:2768345 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PLEK (Pleckstrin). Expected localisation Cytoplasmic and extracellular in marrow and lymph node (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06656-3). Validated IHC antibody: A06656-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Germinal center cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A06656-3); 2. Fixation: Image fixative and duration unreported (datasheet A06656-3); 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 retrieval: EDTA pH 8.0 (datasheet A06656-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06656-3); 8. Primary antibody: Rabbit anti-PLEK, 2-5 μg/ml (datasheet A06656-3); 9. Primary incubation: Overnight at 4 °C (datasheet A06656-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Myeloid peroxidase may raise marrow DAB background (standard IHC practice); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt P08567, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoli., Datasheet A06656-3, PMC11979283, PMC10929796. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PLEK/immunohistochemistry-ihc-guide.