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PKIA / cAMP-dependent protein kinase inhibitor alpha · IHC design guide
Design Immunohistochemistry for PKIA
Plan PKIA paraffin IHC around the reported cytoplasmic and membranous tissue staining (HPA tissue IHC) and the catalog antibody’s 1:100–1:300 dilution (datasheet). Interpret tissue staining cautiously because agreement with RNA expression is very low (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PKIA 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 membranous; tissue pattern uncertain (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+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 | Very low agreement between staining and RNA expression (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; chain spans residues 2–76 (UniProt) |
Section 1
Recommended PKIA IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published PKIA IHC protocol (PMC10466921: immunohistochemistry methods).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A30465) |
| Fixation | Image fixative and duration unreported (datasheet A30465); 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-PKIA, 1:100 - 1:300 (datasheet A30465) |
| 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 | PKIA-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule); the published protocol also uses citrate buffer (PMC10466921: methods).
Section 2
What Is the Expected PKIA Staining Pattern?
PKIA staining is reported in the cytoplasm and at cell membranes across several tissues, including high staining in breast glandular and cervical squamous epithelial cells (HPA: tissue IHC). PKIA has no annotated transmembrane segment, and UniProt assigns no subcellular location (UniProt P61925). Treat this pattern as provisional: HPA rates the tissue IHC evidence Uncertain because antibody staining has very low consistency with RNA expression (HPA: reliability).
| Cytoplasmic staining, with or without a membranous outline, in breast glandular cells or cervical squamous cells. | This matches the reported compartments and high-staining cell types (HPA: tissue IHC). Compare staining with adjacent tissue structures and controls before scoring; the reported pattern has Uncertain reliability (HPA: reliability). |
| Strong staining confined to nuclei, without the reported cytoplasmic or membranous pattern. | A nuclear-only result conflicts with the reported tissue pattern (HPA: tissue IHC). Consider nonspecific signal or a staining artefact; UniProt provides no subcellular annotation that would independently resolve the discrepancy (UniProt P61925). |
| Prominent staining of adipocytes or cardiomyocytes. | These cells were reported as Not detected (HPA: adipose tissue and heart muscle IHC). Check for cross-reactivity or endogenous detection activity using appropriate controls (general IHC practice). An isolated positive result needs independent validation because HPA rates the pattern Uncertain (HPA: reliability). |
| Color spread across many cell types or over tissue spaces, obscuring cell boundaries. | Treat this as background until controls show otherwise (general IHC practice). It cannot establish PKIA localization, especially when the reported compartment pattern and antibody validation are Uncertain (HPA: tissue IHC; HPA042791 IHC). |
| No visible stain in breast glandular cells or placental trophoblastic cells. | Both are reported High, so absence of signal warrants checking assay performance (HPA: breast and placenta IHC). They are provisional comparison tissues, not guaranteed positive controls, because HPA reports Uncertain tissue IHC reliability (HPA: reliability). |
💡Expected PKIA appearanceA plausible positive is predominantly cytoplasmic, sometimes membranous, staining in an HPA High cell population such as breast glandular cells; widespread nuclear-only or cell-independent color is suspect (HPA: tissue IHC and reliability).
| Tissue and cell-type choice (HPA: tissue IHC). | Breast glandular cells and placental trophoblastic cells are reported High; adipocytes and cardiomyocytes are Not detected (HPA: tissue IHC). Use these as provisional contrasts because the tissue pattern has Uncertain reliability (HPA: reliability). |
| Reported compartment versus topology (HPA: tissue IHC; UniProt P61925). | HPA describes cytoplasmic and membranous staining (HPA: tissue IHC). PKIA has no annotated transmembrane segment, so a membrane outline alone does not establish membrane insertion (UniProt P61925 topology). |
| Antibody and tissue-pattern confidence (HPA: HPA042791 IHC; reliability). | HPA042791 is rated Uncertain for IHC; HPA also reports very low consistency between tissue staining and RNA expression (HPA: HPA042791 IHC; reliability). Require controls and independent corroboration for unexpected results (general IHC practice). |
| IF/ICC Q: Is a cellular IF pattern established? (HPA: subcellular). | A: No main location or ICC-IF images are supplied (HPA: subcellular). The tissue IHC pattern does not establish an IF compartment; evaluate IF with its own controls and guide (general IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in an HPA High tissue (HPA: tissue IHC). | The IHC run may have failed, or this uncertain HPA pattern may not reproduce (general IHC practice; HPA: reliability). | Check detection controls and the antibody's IHC-P instructions; compare a second reported High cell population before interpreting absence as biological (general IHC practice; HPA: tissue IHC). |
| Strong nuclear-only signal. | The result disagrees with the reported cytoplasmic and membranous tissue pattern (HPA: tissue IHC). | Inspect negative controls and tissue morphology, then repeat or independently validate before assigning nuclear PKIA (general IHC practice; HPA: reliability). |
| Adipocytes or cardiomyocytes stain prominently. | These cell types are reported Not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Run no-primary and detection controls, assess whether color tracks the cells, and seek independent confirmation before calling them PKIA-positive (general IHC practice). |
| Diffuse chromogen obscures cellular localization. | Excess background or endogenous detection activity may obscure specific staining (general IHC practice). | Review blocking, washing, antibody concentration and detection controls; score only interpretable cellular staining (general IHC practice). |
| Membrane outlines are the only apparent positive signal. | HPA reports membranous staining, but PKIA has no annotated transmembrane segment (HPA: tissue IHC; UniProt P61925 topology). | Check whether cytoplasmic staining and the reported cell-type distribution accompany the outlines; validate isolated membrane staining independently (HPA: tissue IHC; general IHC practice). |
| The slide pattern appears convincing but conflicts with RNA distribution. | HPA reports very low consistency between antibody staining and RNA expression (HPA: reliability). | Record the discrepancy and seek independent evidence before treating the IHC pattern as established PKIA expression (HPA: reliability; general IHC practice). |
Experiment evidence
IHC & IF images showing PKIA staining
A30465 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using IPKA Antibody. The picture on the right is blocked with the syn
A30465 · IFImmunofluorescence analysis of HeLa cells, using IPKA Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000171033-PKIA/tissue/cerebral+cortex#imid_11369419
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000171033-PKIA/tissue/colon#imid_11369604
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000171033-PKIA/tissue/liver#imid_11369528
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000171033-PKIA/tissue/kidney#imid_11369609
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PKIA IHC & IF
🧪Run breast first and score glandular cells for PKIA staining (HPA: High in breast glandular cells); run adipose tissue as the biological negative, with adipocytes expected to lack detectable staining (HPA: Not detected in adipocytes). On the breast slide, examine adjacent nonglandular cells for background staining, but do not assume they are PKIA-negative because their expression is not specified in the supplied HPA row.
Positive control tissue: Breast (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for PKIA; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use knockout material or a peptide-blocked section as a target-specific control (A30465 tissue-IHC caption: synthesized-peptide block). For chromogenic detection, quench endogenous peroxidase and check the breast section for background signal before scoring glandular cells (HPA: High in breast glandular cells).
⚠️Feasibility: A PKIA-specific fixation window and the effect or necessity of antigen retrieval are unreported in the supplied evidence; optimize retrieval on matched paraffin sections. The selected A30465 paraffin-section caption does not report a fixative, so fixation cannot be inferred (A30465 tissue-IHC caption: fixative not stated). The supplied evidence does not establish that frozen sections or IF/ICC are easier, and it documents no breast-specific artefact (HPA subcellular: no ICC-IF images; A30465 tissue-IHC caption: paraffin-embedded brain). The selected A30465 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A30465).
HPA tissue IHC evidence for PKIA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Very low consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PKIA IHC Tips
Troubleshoot PKIA staining in paraffin sections by checking retrieval, tissue context and compartment pattern before scoring chromogenic signal.
What retrieval should I try first when PKIA staining is weak in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced antigen retrieval at 95–98 °C for 20 minutes (page retrieval setting). Let sections cool consistently, then compare the same tissue across runs with matched antibody incubation and chromogen development (standard IHC practice). If staining remains weak, test an alternative retrieval buffer on adjacent sections while watching for tissue damage and increased background (standard IHC practice). PKIA has no transmembrane segment, but its antibody epitope is unspecified, so topology alone cannot establish which retrieval condition exposes it best (UniProt P61925 topology; supplied antibody evidence).
Could fixation explain weak or uneven PKIA staining?
PKIA-specific sensitivity to fixation is unknown because no target-specific fixation evidence is supplied (supplied evidence). The catalog image shows staining in paraffin-embedded human brain and peptide blocking, but its caption does not report the fixative (A30465 caption). For a troubleshooting comparison, process adjacent sections with the same retrieval, antibody incubation and detection conditions, and record their known fixation histories (standard IHC practice). Excessive or uneven fixation can alter epitope access in IHC generally; assess that possibility alongside section thickness, drying and tissue preservation without assigning a PKIA-specific effect (standard IHC practice).
How should I assess a PKIA staining pattern that appears membrane associated?
Compare the chromogenic signal with cell boundaries and cytoplasm at high magnification, using the counterstain to identify individual cells (standard IHC practice). Tissue IHC describes cytoplasmic and membranous PKIA staining, although the reported staining has uncertain reliability (HPA tissue IHC). PKIA has no transmembrane segment, and UniProt provides no subcellular annotation; a sharp membrane rim therefore warrants careful validation rather than automatic acceptance (UniProt P61925 topology and subcellular record). Check whether the pattern persists in intact tissue away from cut edges and whether peptide blocking reduces signal in the illustrated tissue context (standard IHC practice; A30465 caption).
Could an uncharacterised epitope account for inconsistent PKIA staining?
The supplied record lists 0 isoforms and no annotated domains, so it provides no basis for assigning inconsistent staining to a particular isoform (UniProt P61925 record). PKIA is a 76-amino-acid protein with an N-acetylthreonine at position 2, but the catalog antibody’s recognized epitope is not supplied (UniProt P61925 record; supplied antibody evidence). Do not infer that this modification blocks binding without epitope information or a direct comparison (standard IHC interpretation). Compare adjacent sections using the same retrieval and detection settings, and interpret peptide-block reduction as supportive evidence for antibody binding, not proof of exclusive PKIA recognition (standard IHC practice; A30465 caption).
How can I investigate PKIA localisation with multiplex immunofluorescence?
Treat IF as a separate validation experiment because the supplied PKIA subcellular record includes no ICC/IF images (HPA subcellular record). Pair PKIA with a marker for the cell type under study; a glandular epithelial marker is a reasonable choice where glandular cells show high tissue IHC staining (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores, consider a far-red PKIA channel if tissue autofluorescence is strong, and collect unstained and single-label controls (standard IF practice). Because PKIA has no transmembrane segment and the epitope is unspecified, test gentle permeabilisation empirically rather than assuming which side of a membrane must be accessed (UniProt P61925 topology; supplied antibody evidence).
How do I reduce diffuse or punctate background in PKIA chromogenic IHC?
Examine a no-primary control to distinguish secondary-reagent and tissue background from antibody-dependent staining (standard IHC practice). Include an endogenous peroxidase block before peroxidase-based DAB detection, and adjust blocking, antibody concentration and wash stringency one variable at a time (standard IHC practice). Inspect edges, folds and damaged areas before changing retrieval, since these can concentrate chromogen or disrupt tissue morphology (standard IHC practice). The catalog brain image includes a synthesized-peptide blocking panel, but that comparison alone does not establish that every stained structure represents PKIA (A30465 caption; standard IHC interpretation). Record exposure to chromogen and counterstain consistently when comparing sections (standard IHC practice).
What is a defensible way to quantify PKIA staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then keep retrieval, detection and image acquisition consistent across sections (standard IHC practice). For cell-based analysis, report the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories 0–3 with a possible range of 0–300 (standard IHC scoring practice). Normalise positive-cell counts to all evaluable cells of the specified type, or report positive-cell density per mm² of viable tissue (standard IHC scoring practice). Report compartments separately because tissue IHC describes both cytoplasmic and membranous staining, and flag its uncertain reliability when interpreting differences (HPA tissue IHC).
How can I distinguish plausible PKIA staining from artefact?
Prioritise staining within intact cells and compare its compartment pattern with the reported cytoplasmic and membranous tissue profile, while remembering that this profile has uncertain reliability (HPA tissue IHC). Compare cell types within the same section: high staining is reported in several glandular populations, whereas adipocytes are reported as not detected (HPA tissue IHC). Treat staining limited to section edges, folds or necrotic regions as suspect, and use a no-primary control to assess endogenous enzyme or detection background (standard IHC practice). Peptide blocking reduced the illustrated brain signal, but the paraffin-section caption does not establish fixation conditions or independently prove target specificity (A30465 caption; standard IHC interpretation).
Boster reagents
Best PKIA / cAMP-dependent protein kinase inhibitor alpha IHC Antibodies
Anti-PKIA A30465 has IHC data from paraffin-embedded human brain tissue and IF data from HeLa cells (A30465 image captions); listed reactivity covers human, mouse and rat (catalog: reactivity).
Real IHC data
Anti-IPKA PKIA Antibody
Cat # A30465
A30465 will render with its IHC image of paraffin-embedded human brain tissue and a peptide-blocked comparison (A30465 IHC caption). Its separate IF image shows HeLa cells with a peptide-blocked comparison (A30465 IF caption); the catalog lists IHC, IF and ICC applications (catalog: applications).
Which to pick: Choose A30465 for paraffin-section tissue IHC based on its own human brain IHC image; the fixative is unreported (A30465 IHC caption). For IF/ICC, A30465 lists both applications and provides an IF image in HeLa cells (catalog: applications; A30465 IF caption). For cross-species planning, A30465 is a rabbit polyclonal antibody listed as reactive with human, mouse and rat; its supplied IHC image shows human tissue (catalog: host, clonality and reactivity; A30465 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P61925 (IPKA_HUMAN, cAMP-dependent protein kinase inhibitor alpha).
- Human Protein Atlas. PKIA tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. PKIA subcellular location (ICC-IF): Highest expression in HUVEC/TERT2: 95.9 nTPM.
- Human Protein Atlas. PKIA antibody validation summary (1 antibodies).
- Exploring the methylation status of CFTR and PKIA genes as potential biomarkers for lung adenocarcinoma. Orphanet journal of rare diseases 2023 — PMC10466921.
- PubMed PMID:1770951 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:22814378 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PKIA (cAMP-dependent protein kinase inhibitor alpha). Expected localisation Cytoplasmic and membranous; tissue pattern uncertain (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A30465 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A30465); 2. Fixation: Image fixative and duration unreported (datasheet A30465); 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-PKIA, 1:100 - 1:300 (datasheet A30465); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Very low agreement between staining and RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms annotated; chain spans residues 2–76 (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt P61925, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in HUVEC/TERT2: 95.9 nTPM, Datasheet A30465, PMC10466921. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PKIA/immunohistochemistry-ihc-guide.