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PPP1R14D / Protein phosphatase 1 regulatory subunit 14D · IHC design guide
Design Immunohistochemistry for PPP1R14D
This paraffin-section IHC guide covers PPP1R14D’s expected cytoplasmic location (UniProt) and reported glandular-cell staining in colon, rectum and small intestine (HPA tissue IHC). It gives the catalog antibody’s IHC dilution of 1:100–1:300 (datasheet A14433) and flags the uncertain specificity of the tissue staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PPP1R14D 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 tissue staining; reliability uncertain (HPA tissue IHC) | |
| Staining pattern | Glandular-cell cytoplasm in gastrointestinal tissues (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A14433) | |
| Positive control | Colon+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 paraffin-section fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target binding; staining needs verification (HPA tissue IHC) | |
| Regulation | Expression regulation not established (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–145 chain (UniProt) |
Section 1
Recommended PPP1R14D IHC & IF Protocols
The catalog antibody’s IHC-P protocol and one published PPP1R14D tissue IHC protocol are available for comparison (datasheet A14433; PMC9635868).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A14433) |
| Fixation | Image fixative and duration unreported (datasheet A14433); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A14433); 20 min, 95–100 °C (standard) |
| 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-PPP1R14D, 1:100-1:300 (datasheet A14433) |
| 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 | PPP1R14D-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, including the gastrointestinal tract. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA, pH 9.0, for the catalog antibody (datasheet A14433); the published protocol used heated citrate, pH 6.0 (PMC9635868).
Section 2
What Is the Expected PPP1R14D Staining Pattern?
PPP1R14D is annotated as cytoplasmic and has no transmembrane segment (UniProt Q9NXH3: subcellular location and topology). In paraffin-section IHC, the strongest reported staining is in glandular cells of the colon, rectum and small intestine (HPA: High). Interpret this pattern cautiously: HPA rates tissue IHC reliability as Uncertain because presumed off-target binding was observed and disregarded, pending external verification (HPA: reliability description).
| Cytoplasmic staining in gastrointestinal glandular cells. | This matches the annotated compartment (UniProt Q9NXH3: Cytoplasm) and HPA's reported High staining in colon, rectum and small-intestine glandular cells (HPA: tissue IHC). It is a pattern match, not independent proof of antibody specificity (HPA: Uncertain reliability). |
| Predominantly nuclear or sharply membrane-restricted staining. | This conflicts with the cytoplasmic annotation and lack of a transmembrane segment (UniProt Q9NXH3: location and topology). Check controls and repeatability before calling it PPP1R14D; HPA's uncertain IHC validation increases the need for caution (HPA: reliability). |
| Strong staining in a cell type reported as not detected. | For example, HPA reports adipocytes in adipose tissue and bronchial respiratory epithelial cells as Not detected (HPA: tissue IHC). Unexpected staining may reflect cross-reactivity or endogenous chromogen-generating activity; compare matched controls before assigning a cause. |
| Diffuse staining across cells and extracellular areas. | A widespread haze does not fit HPA's cell-associated cytoplasmic profile (HPA: tissue IHC). In general IHC practice, assess nonspecific reagent binding, incomplete washing and endogenous detection activity with appropriate controls. |
| No stain in an expected positive section. | Colon, rectum and small-intestine glandular cells are reported High (HPA: tissue IHC). A blank section warrants a check of section quality, retrieval, antibody conditions and detection controls; HPA's Uncertain rating means the reported pattern is a provisional benchmark (HPA: reliability). |
💡Expected PPP1R14D appearanceCall a provisional positive when glandular cells show cytoplasmic staining, strongest in colon, rectum or small intestine (UniProt Q9NXH3: Cytoplasm; HPA: High); dominant nuclear, membrane-restricted or widespread acellular color is suspect (UniProt Q9NXH3: topology; HPA: tissue IHC).
| Tissue and cell selection | Colon, rectum and small-intestine glandular cells have High reported staining; appendix, duodenum and gallbladder glandular cells, and kidney tubular cells, have Medium staining (HPA: tissue IHC). Choose comparisons at the cell level. |
| Low and undetected comparators | Stomach glandular cells and smooth-muscle cells are Low, while adipocytes in adipose tissue are Not detected (HPA: tissue IHC). These labels describe observed IHC signal, not an absolute protein absence or a validated specificity test. |
| Antibody evidence | The listed antibody HPA041846 is rated Uncertain for IHC; HPA reports presumed off-target binding and pending external verification (HPA: antibody IHC status and reliability description). Treat even plausible positive staining as provisional. |
| Compartment and topology | A cytoplasmic pattern is consistent with UniProt's location annotation; no transmembrane segment is annotated (UniProt Q9NXH3: location and topology). A crisp membrane-only pattern lacks support from this record. |
| Protein state | PPP1R14D inhibits PPP1CA only when phosphorylated, and Thr58 is annotated as a phosphothreonine site (UniProt Q9NXH3: function and modified residues). The payload gives no antibody epitope or phospho-specificity, so staining cannot establish phosphorylation state. |
| Processing and isoforms | UniProt lists one 1–145 chain, no signal peptide or propeptide, and no annotated isoforms (UniProt Q9NXH3: processing and isoforms). Do not assign a separate staining compartment or cell pattern to an unreported processed form. |
| IF/ICC Q&A | Can IF/ICC confirm the compartment here? HPA provides no main ICC-IF location or cell-line images, and the listed antibody has no ICC validation entry (HPA: subcellular and antibody records). This section therefore offers no IF/ICC protocol or image-based confirmation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected gastrointestinal glandular cells are blank. | A technical failure is possible; HPA reports these cells as High but rates tissue IHC Uncertain (HPA: tissue IHC and reliability). | In general IHC practice, check tissue integrity, retrieval, primary-antibody setup and detection reagents with a known working control; repeat before interpreting a negative result. |
| Only nuclei stain strongly. | The observed compartment conflicts with UniProt's cytoplasmic annotation (UniProt Q9NXH3: subcellular location). Nonspecific staining or interpretation error is possible. | Compare a primary-omission control and cell morphology, then repeat under adjusted general IHC blocking and washing conditions; avoid scoring nuclei as a supported PPP1R14D pattern. |
| A crisp cell-surface rim dominates. | UniProt annotates no transmembrane segment and places PPP1R14D in the cytoplasm (UniProt Q9NXH3: topology and location). | Recheck the section's compartment boundaries and staining controls. Require independent specificity evidence before assigning a membrane-localized result to PPP1R14D. |
| Unexpected cells stain as strongly as the positive comparator. | Cross-reactivity is plausible because presumed off-target binding was observed (HPA: Uncertain reliability); endogenous chromogenic activity is another general IHC possibility. | Compare a matched primary-omission control and, where applicable, an endogenous-enzyme control; document the precise cell type rather than treating all tissue staining as equivalent. |
| The whole section has a brown haze. | Diffuse background can arise from general IHC issues such as nonspecific binding, insufficient washing or endogenous detection activity; HPA describes a cytoplasmic cellular profile (HPA: tissue IHC). | Use a matched negative control, review blocking and wash steps, and inspect signal at the cell level before scoring intensity. |
| Smooth muscle stains weakly despite the annotated role in contraction. | UniProt describes a phosphorylation-dependent function, while HPA reports Low staining in smooth-muscle cells (UniProt Q9NXH3: function; HPA: tissue IHC). Function does not predict strong IHC signal. | Score the observed cells against the HPA tissue pattern and controls; do not increase exposure or development solely to force a strong smooth-muscle result. |
Experiment evidence
IHC & IF images showing PPP1R14D staining
A14433 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Tris-EDTA, pH9.0 was used for antigen retrieval. 2 Antibody was diluted a
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166143-PPP1R14D/tissue/cerebral+cortex#imid_10752003
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166143-PPP1R14D/tissue/colon#imid_10751937
HPA · RectumHuman Protein Atlas IHC, Rectum. Source: https://www.proteinatlas.org/ENSG00000166143-PPP1R14D/tissue/rectum#imid_10751905
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166143-PPP1R14D/tissue/liver#imid_10751922
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PPP1R14D IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use bronchus respiratory epithelial cells as the negative tissue (HPA: Not detected); on the colon slide, compare glandular staining with adjacent nonglandular stroma as an internal background check, without assuming the stroma is antigen negative (HPA: signal assigned to glandular cells).
Positive control tissue: Colon (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 PPP1R14D; 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 clonality (standard IHC practice). Use knockout tissue or a validated peptide block as a biological negative, and quench endogenous peroxidase before chromogenic detection in colon sections (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A14433 paraffin-section caption does not state a fixative (A14433 caption: fixative unreported). The caption reports Tris-EDTA retrieval at pH 9.0 and primary antibody at 1:200 overnight at 4°C, but does not establish that retrieval is required (A14433 caption). The supplied evidence does not establish that frozen-section IHC or IF/ICC is easier; in colon, inspect mucus and luminal debris for trapped chromogen (standard IHC practice).
HPA tissue IHC evidence for PPP1R14D
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Presumed off target binding observed and disregarded. 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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PPP1R14D IHC Tips
Troubleshoot PPP1R14D staining in paraffin section IHC using the catalog image, protein record, and tissue patterns as distinct lines of evidence.
What retrieval should I try first for weak PPP1R14D staining in paraffin sections?
Start with heat-mediated Tris-EDTA retrieval at pH 9.0 for the catalog antibody (datasheet A14433). The selected paraffin-section image used that buffer, a 1:200 primary dilution overnight at 4 °C, and a 1:200 secondary for 45 minutes at room temperature (caption A14433). If staining is weak, compare carefully controlled heating and cooling conditions before changing antibody concentration (standard IHC practice). Keep a colon or small-intestine glandular-cell control alongside the test section, while treating its expected staining as provisional because the tissue evidence has uncertain reliability (HPA: high in glandular cells; HPA: uncertain reliability). Assess background and tissue damage with each retrieval change (standard IHC practice).
Could fixation explain lost PPP1R14D staining?
Target-specific fixation sensitivity is unknown: the selected image identifies paraffin-embedded tonsil but does not state its fixative (caption A14433). Record the fixative, fixation duration, tissue thickness, and processing history for each case before comparing staining intensity (standard IHC practice). Test a shared, consistently processed positive-control section across runs and inspect morphology for underfixation or excessive processing effects (standard IHC practice). Maintain the documented Tris-EDTA pH 9.0 retrieval and 1:200 primary dilution while evaluating fixation variables (datasheet A14433; caption A14433). Do not assign a fixation cause solely from a negative section: the reported tissue staining has uncertain reliability (HPA: uncertain reliability).
Where should convincing PPP1R14D staining appear?
Prioritize cytoplasmic staining within identifiable cells because PPP1R14D is annotated as cytoplasmic and has no transmembrane segment (UniProt Q9NXH3: subcellular location and topology). Colon, rectum, and small-intestine glandular cells are reported as high-staining reference populations, although the tissue assessment is uncertain (HPA: positive tissues; HPA: uncertain reliability). Compare the same compartment and cell population on each section, using a matched negative control to judge diffuse chromogen deposits (standard IHC practice). Predominantly nuclear or sharply membrane-restricted staining should trigger a specificity check against the expected cytoplasmic pattern (UniProt Q9NXH3: cytoplasm and topology; standard IHC practice). Preserve morphology with a restrained counterstain so that glandular cells remain identifiable (standard IHC practice).
How should I assess epitope-dependent or phosphorylation-dependent staining?
The record annotates one 145-amino-acid chain, no isoforms, and phosphothreonine at residue 58 (UniProt Q9NXH3: processing, isoforms, modified residues). Those annotations do not establish which sequence the catalog antibody recognizes or whether phosphorylation changes its binding (UniProt Q9NXH3: protein record; caption A14433: epitope not reported). Request or inspect antibody epitope information before interpreting differential staining as a change in protein abundance or phosphorylation (standard IHC practice). Run serial sections with the same retrieval and detection settings, and compare a second independently characterized antibody if available (standard IHC practice). Phosphorylation is relevant to PPP1R14D inhibitory activity, but total-protein IHC alone cannot establish that activity (UniProt Q9NXH3: function; standard IHC interpretation).
How can I check a PPP1R14D signal by multiplex IF?
Use IF as a separate validation experiment; the selected antibody image documents paraffin-section chromogenic staining, with no IF result supplied (caption A14433). Multiplex PPP1R14D with an independently validated glandular-cell marker in intestinal tissue, where glandular staining is reported, and include single-label controls for each channel (HPA: intestinal glandular cells; standard IF practice). Choose fluorophores after measuring tissue autofluorescence and favor a well-separated far-red channel if background is strongest at shorter wavelengths (standard IF practice). Because PPP1R14D is cytoplasmic with no transmembrane segment, optimize mild permeabilisation to access intracellular epitopes while preserving morphology (UniProt Q9NXH3: cytoplasm and topology; standard IF practice). Confirm channel separation before interpreting apparent colocalisation (standard IF practice).
What should I change when PPP1R14D chromogen covers the section?
First compare a no-primary control and a matched tissue section to distinguish antibody-associated staining from detection background (standard IHC practice). For peroxidase-based chromogenic IHC, verify the peroxidase block and shorten chromogen development if the no-primary control also darkens (standard IHC practice). The selected image used a 1:200 primary overnight at 4 °C and a 1:200 secondary for 45 minutes at room temperature; treat these as its reported conditions, not universal optima (caption A14433). Titrate the primary and secondary separately, adjust blocking and washes, and check section drying or edge deposition (standard IHC practice). Reassess cytoplasmic staining in intact glandular cells after background falls (UniProt Q9NXH3: cytoplasm; HPA: intestinal glandular cells).
How should I score PPP1R14D across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, since PPP1R14D is annotated as cytoplasmic and reported intestinal signal is glandular (UniProt Q9NXH3: cytoplasm; HPA: intestinal glandular cells). For chromogenic sections, record the percentage of positive target cells and intensity categories, then calculate an H-score if the study needs a combined measure (standard IHC practice). Normalize stained-cell counts to the number of evaluable cells, or area-based counts to evaluable tissue area in mm²; exclude folds and necrosis consistently (standard IHC practice). Hold retrieval, exposure to chromogen, counterstain, and image-analysis thresholds constant across batches (standard IHC practice). Report the uncertain tissue-reference reliability when interpreting between-tissue differences (HPA: uncertain reliability).
How do I distinguish true PPP1R14D staining from artefact?
A plausible signal is cellular and cytoplasmic, particularly in intestinal glandular cells reported as positive (UniProt Q9NXH3: cytoplasm; HPA: colon, rectum, and small-intestine glandular cells). Treat predominantly nuclear or membrane-only staining, staining limited to damaged edges or necrosis, and signal in a no-primary control as prompts to investigate artefact (UniProt Q9NXH3: cytoplasm and topology; standard IHC practice). Check whether peroxidase blocking and chromogen timing explain deposits outside intact cells (standard IHC practice). Use matched controls and, when possible, independent antibody validation before assigning an unexpected cell population as positive (standard IHC practice). Tissue-reference staining remains provisional because presumed off-target binding was observed and external verification is pending (HPA: uncertain reliability).
Boster reagents
Best PPP1R14D / Protein phosphatase 1 regulatory subunit 14D IHC Antibodies
A14433 has IHC data from paraffin-embedded human tonsil (IHC image caption); its listed reactivity covers human, mouse and rat, and IF is listed without an IF image (catalog).
Real IHC data
Anti-PPP1R14D/Gbpi 1 Antibody
Cat # A14433
A14433 will render with an IHC image of paraffin-embedded human tonsil (IHC image caption). Its listed applications include IHC and IF, and its listed reactivity includes human, mouse and rat (catalog).
Which to pick: For tissue IHC, choose A14433: its own image shows paraffin-embedded human tonsil with Tris-EDTA retrieval at pH 9.0 and primary antibody at 1:200 (IHC image caption); the fixative is unreported (IHC image caption). For IF, A14433 is listed at 1:50, but no IF image is supplied, and ICC is not listed (catalog). A14433 is polyclonal and lists mouse and rat reactivity alongside human; the supplied IHC image documents human tissue only (catalog; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NXH3 (PP14D_HUMAN, Protein phosphatase 1 regulatory subunit 14D).
- Human Protein Atlas. PPP1R14D tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. PPP1R14D subcellular location (ICC-IF): Highest expression in RT-4: 1.8 nTPM.
- Human Protein Atlas. PPP1R14D antibody validation summary (1 antibodies).
- PPP1R14D promotes the proliferation, migration and invasion of lung adenocarcinoma via the PKCα/BRAF/MEK/ERK signaling pathway. International journal of oncology 2022 — PMC9635868.
- PubMed PMID:12974676 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PPP1R14D (Protein phosphatase 1 regulatory subunit 14D). Expected localisation Cytoplasmic tissue staining; reliability uncertain (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A14433). Validated IHC antibody: A14433 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A14433); 2. Fixation: Image fixative and duration unreported (datasheet A14433); 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: Tris-EDTA pH 9.0 (datasheet A14433); 20 min, 95–100 °C (standard); 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-PPP1R14D, 1:100-1:300 (datasheet A14433); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific); Caveat: Presumed off-target binding; staining needs verification (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–145 chain (UniProt); Regulation: Expression regulation not established (UniProt). Evidence: UniProt Q9NXH3, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in RT-4: 1.8 nTPM, Datasheet A14433, PMC9635868. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PPP1R14D/immunohistochemistry-ihc-guide.