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POLB / DNA polymerase beta · IHC design guide
Design Immunohistochemistry for POLB
Plan POLB chromogenic IHC in paraffin sections using the general nuclear tissue pattern reported by HPA (HPA tissue IHC). Compare high staining in breast glandular cells with undetected staining in adipocytes, and evaluate the IHC-validated antibody A01946-2 at 1:50–1:200 (HPA tissue IHC; datasheet: IHC dilution).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
POLB 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 | General nuclear expression in tissue (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 | Breast+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01946-2) | |
| Caveat | DNA damage can shift POLB into nuclei (UniProt) | |
| Regulation | DNA damage changes nuclear localisation (UniProt) | |
| Isoform / epitope | One 1–335 chain; no isoforms annotated (UniProt) |
Section 1
Recommended POLB IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published POLB staining methods from four articles (PMC9869049; PMC7957714; PMC13232957; PMC4964131).
| Sample | Paraffin-embedded rat lung tissue; fixative not specified (datasheet A01946-2) |
| Fixation | Image fixative and duration unreported (datasheet A01946-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-POLB, 1:50-1:200 (datasheet A01946-2) |
| 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 | POLB-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: General 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 antigen retrieval); citrate pH 6 is a published alternative (PMC9869049).
Section 2
What Is the Expected POLB Staining Pattern?
POLB has general nuclear expression in tissue IHC (HPA: tissue IHC, Enhanced reliability). Strong staining is reported in breast, gallbladder and salivary glandular cells, testicular round or early spermatids, tonsillar squamous epithelium and bladder urothelium (HPA: High). Cytoplasmic signal can occur under normal conditions, with nuclear translocation after DNA damage (UniProt P06746: subcellular location). POLB has no transmembrane segment (UniProt P06746: topology).
| Clear nuclear staining in sampled glandular or urothelial cells (HPA: general nuclear expression; High in breast glandular cells and bladder urothelial cells). | This fits the reported tissue pattern when the stained cells are identifiable on the section (HPA: tissue IHC). Compare intensity within the same run: HPA levels describe its observations, not a universal staining threshold (HPA: tissue IHC). |
| Predominantly membrane-rim or extracellular staining, with little signal in the expected cells (HPA: general nuclear expression). | Question the pattern: POLB has no transmembrane segment, and membrane or extracellular localization is not listed (UniProt P06746: topology and subcellular location). Check staining controls and tissue morphology before assigning that signal to POLB (standard IHC practice). |
| Strong staining in adipocytes, cardiomyocytes or smooth muscle cells, while expected positive cells are also present (HPA: Not detected in those cell types). | Treat this as a specificity warning, not proof of cross-reactivity: HPA reports those cells as not detected in its tissue IHC data (HPA: tissue IHC). Compare a no-primary control for endogenous detection activity and review cell identity (standard IHC practice). |
| Diffuse staining covers cells and intervening tissue, so nuclei cannot be scored (HPA: general nuclear expression). | This cannot establish POLB localization. Nonspecific binding or detection background may obscure the cellular pattern (standard IHC practice). Review the no-primary control, blocking and antibody concentration before interpreting intensity (standard IHC practice). |
| No signal in a section containing identifiable breast glandular cells or bladder urothelial cells (HPA: High in those cells). | Investigate assay performance and sampling first; an unstained expected positive does not establish biological absence (standard IHC practice). HPA's High designation supports these as reference cell populations but does not guarantee every specimen or staining run (HPA: tissue IHC). |
💡Expected POLB appearanceCall a convincing positive when identifiable glandular, urothelial or other HPA-High cells show clear nuclear staining, with possible cytoplasmic signal (HPA: tissue IHC; UniProt P06746: subcellular location); isolated membrane-rim or diffuse tissue-wide signal is suspect (UniProt P06746: topology; standard IHC practice).
| Which cells provide a reference? | HPA reports High staining in breast, gallbladder and salivary glandular cells; round or early spermatids; tonsillar squamous epithelial cells; and urothelial cells (HPA: tissue IHC). Adipocytes, cardiomyocytes and smooth muscle cells are reported as Not detected, useful for comparison within their sampled tissues (HPA: tissue IHC). |
| How much confidence does the tissue profile carry? | HPA rates its tissue IHC profile Enhanced, describing high consistency between antibody staining and RNA expression (HPA: tissue IHC reliability). HPA049104 and CAB011616 have Enhanced IHC validation; HPA069013 is Supported (HPA: antibody validation). Validation applies to those antibodies and assays, not automatically to another catalog antibody (standard IHC practice). |
| Does POLB biology predict surface staining or shedding? | No signal peptide, propeptide or transmembrane segment is annotated; the protein is listed as one chain spanning residues 1–335 (UniProt P06746: processing and topology). These annotations give no basis for an expected membrane-surface or shed-product pattern in tissue IHC (UniProt P06746: processing and topology). |
| Can nuclear and cytoplasmic results differ? | UniProt lists cytoplasmic localization under normal conditions and nuclear translocation following DNA damage (UniProt P06746: subcellular location). HPA describes general nuclear expression in tissue IHC (HPA: tissue IHC). Record both compartments and the specimen context; the supplied sources do not establish a fixed nuclear-to-cytoplasmic scoring cutoff. |
| What about IF/ICC? | HPA's ICC-IF summary reports vesicles and cytosol; vesicles are Approved and cytosol Supported (HPA: subcellular ICC-IF). Those observations answer the localization question for that assay. They do not replace the nuclear tissue-IHC profile or supply an IF/ICC protocol (HPA: subcellular ICC-IF; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are unstained (HPA: High in breast glandular cells or bladder urothelial cells). | The relevant cell population may be absent from the viewed area, or an IHC step may have failed (standard IHC practice). | Confirm cell identity and run controls; check the antibody's IHC validation, retrieval conditions and detection reagents for this run (HPA: antibody validation; standard IHC practice). |
| Brown signal appears throughout tissue, including areas without identifiable cells (standard IHC practice). | Detection background or nonspecific binding can conceal localization (standard IHC practice). | Inspect a no-primary control, review blocking and detection steps, then adjust antibody concentration using the catalog antibody's IHC instructions (standard IHC practice). |
| Adipocytes, cardiomyocytes or smooth muscle cells appear strongly positive (HPA: Not detected). | Cell misidentification, nonspecific staining or endogenous detection activity are possible; HPA's result alone cannot distinguish them (HPA: tissue IHC; standard IHC practice). | Recheck morphology alongside an expected positive population and compare a no-primary control (HPA: tissue IHC; standard IHC practice). |
| Signal seems confined to cell borders (HPA: general nuclear expression). | A border-only pattern conflicts with the reported tissue localization and lack of a transmembrane segment (HPA: tissue IHC; UniProt P06746: topology). | Check focus, counterstain and negative control, then score nuclear and cytoplasmic signal separately (standard IHC practice; UniProt P06746: subcellular location). |
| Cytoplasmic staining exceeds nuclear staining in a tissue section (HPA: general nuclear expression). | Cytoplasmic POLB is biologically plausible, while tissue IHC usually shows general nuclear expression (UniProt P06746: subcellular location; HPA: tissue IHC). | Document the compartment balance and specimen context; compare an expected positive cell population before calling the result aberrant (HPA: tissue IHC; standard IHC practice). |
| Nuclei cannot be distinguished well enough to score (HPA: general nuclear expression). | Overlapping detection signal or counterstain can hide compartment boundaries (standard IHC practice). | Review an adjacent morphology section and adjust detection or counterstain conditions before assigning nuclear localization (standard IHC practice). |
Experiment evidence
IHC & IF images showing POLB staining
A01946-2 · IHCImmunohistochemical analysis of paraffin-embedded rat lung tissue using anti-DNA Polymerase beta antibody. Counter stained with hematoxylin.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000070501-POLB/tissue/cerebral+cortex#imid_2972908
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000070501-POLB/tissue/cerebellum#imid_2972895
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000070501-POLB/tissue/colon#imid_2972818
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000070501-POLB/tissue/liver#imid_2972819
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049104; green = POLB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049104; green = POLB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for POLB IHC & IF
🧪Run breast first: glandular cells should stain (HPA: Breast glandular cells High); use adipose tissue as the negative, where adipocytes are not detected (HPA: Adipocytes Not detected). On the breast slide, cells used as internal negatives should show only background chromogen; the supplied HPA row does not identify a specific negative breast cell type (HPA: Breast glandular cells High).
Positive control tissue: Breast (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show POLB in A-431, SK-MEL-30, U-251MG, U2OS, THP-1, NIH 3T3, with annotated localisation: Vesicles (approved), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and nonimmune IgG matched to the primary antibody’s host species and immunoglobulin class, with a preparation appropriate to its clonality; use POLB knockout material if available, or a peptide block, to assess specificity (standard IHC practice). Block endogenous peroxidase and check background in the breast glandular tissue before scoring chromogenic staining (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section rat lung caption does not report a fixative (A01946-2 caption). Retrieval dependence is unreported; optimize antigen retrieval empirically for paraffin IHC, and do not assume frozen sections or IF are easier from the supplied evidence (standard IHC practice; HPA: Breast glandular cells High). No breast-specific artefact is documented in the supplied evidence; use the background controls to assess glandular staining.
HPA tissue IHC evidence for POLB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Round or early spermatids | High | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Section 3
Advanced POLB IHC Tips
Troubleshoot POLB staining in paraffin sections by checking retrieval, cell identity, compartment, controls, and scoring before interpreting biological differences.
How should I retrieve POLB in paraffin sections when staining is weak?
Start POLB IHC with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page protocol: A01946-2). Allow slides to cool in retrieval buffer, then wash consistently before applying the IHC-validated antibody (standard IHC practice). Compare a positive tissue section across retrieval runs; breast glandular cells show high POLB staining in the reference profile (HPA: High in breast glandular cells). If staining remains weak, test citrate at pH 6.0 as a fallback on matched sections (standard IHC practice). Keep antibody concentration, detection, and exposure to chromogen constant while comparing retrieval conditions (standard IHC practice).
Does fixation change POLB staining in paraffin sections?
Target-specific fixation sensitivity is unknown here: the selected paraffin-section image identifies rat lung but does not state its fixative (caption: A01946-2). Record fixative, fixation duration, tissue thickness, and processing history before comparing slides from different batches (standard IHC practice). For an initial comparison, process matched specimens with the same fixation and retrieval conditions, then assess morphology alongside POLB signal (standard IHC practice). Avoid attributing weak staining to overfixation without matched evidence, because the supplied POLB IHC evidence does not test fixation effects (caption: A01946-2). Use the page's pH 9.0, 20 min retrieval consistently during that comparison (page protocol: A01946-2).
Should convincing POLB IHC signal be nuclear or cytoplasmic?
Assess nuclear staining first, because the tissue IHC profile describes general nuclear POLB expression (HPA: tissue IHC profile). Cytoplasmic staining can also be biologically plausible: POLB is cytoplasmic under normal conditions and translocates to the nucleus after DNA damage (UniProt P06746: subcellular location). Interpret each compartment separately, using hematoxylin to identify nuclei and tissue architecture in chromogenic sections (standard IHC practice; caption: A01946-2). The IF/ICC subcellular profile reports vesicles and cytosol, so discordance between assays deserves investigation rather than automatic rejection (HPA: subcellular profile). Compare compartment patterns within the same cell type and processing batch before assigning a biological change (standard IHC practice).
Could an isoform or modified epitope explain variable POLB staining?
The supplied record lists one 335-aa POLB chain and no annotated isoforms, so an isoform explanation has no support here (UniProt P06746: processing; isoforms). It reports modified residues at positions 72, 83, and 152, but supplies no antibody epitope or evidence that these modifications alter binding (UniProt P06746: modified residues). Check the catalog antibody's epitope information before proposing masking of a specific site (standard IHC practice). Compare staining across matched retrieval conditions and use a validated positive tissue control to assess reproducibility (standard IHC practice; HPA: High in breast glandular cells). Do not interpret staining intensity as a particular POLB modification state without modification-specific validation (standard IHC practice).
How can I compare POLB IHC with multiplex IF?
Treat IF/ICC as a separate assay when comparing its POLB pattern with chromogenic IHC (standard IHC/IF practice). In breast sections, multiplex with an epithelial marker such as pan-cytokeratin to identify glandular cells, which have high POLB staining in tissue IHC (standard IF practice; HPA: High in breast glandular cells). Choose a far-red fluorophore when shorter-wavelength tissue autofluorescence obscures signal, and include single-stain controls for spectral overlap (standard IF practice). For intracellular nuclear or cytosolic POLB, optimise permeabilisation so antibody reaches the relevant compartment; POLB has no transmembrane segment (UniProt P06746: subcellular location; topology). Evaluate IF and IHC localisation independently because the reported assay profiles differ (HPA: tissue IHC and subcellular profiles).
How do I distinguish POLB staining from chromogenic background?
Run a no-primary control and inspect it alongside the POLB slide for detection-system signal (standard IHC practice). For peroxidase detection, test an endogenous-peroxidase block such as 3% hydrogen peroxide for 10 min, then wash before antibody incubation (standard IHC practice). Titrate the IHC-validated antibody and chromogen development on matched sections if diffuse brown signal obscures cell boundaries (standard IHC practice). Use morphology and the hematoxylin counterstain to separate pigment, tissue folds, and edge staining from cell-associated signal (standard IHC practice; caption: A01946-2). Low staining in lung alveolar cells is reported, so weak local signal alone is inconclusive (HPA: Low in lung alveolar cells).
How should I quantify nuclear and cytoplasmic POLB IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC describes general nuclear expression while POLB can also occupy the cytoplasm (HPA: tissue IHC profile; UniProt P06746: subcellular location). For nuclear staining, record the percentage of positive cells and an H-score from 0–300 using intensity categories 0–3 (standard IHC practice). Score cytoplasmic intensity separately, and report whether counts cover whole tissue or selected regions (standard IHC practice). Normalise positive-cell counts to the number of evaluable cells of the same type, or report density per mm² of viable tissue (standard IHC practice). Apply identical thresholds and exclude folds and necrotic areas consistently (standard IHC practice).
What makes a POLB staining pattern credible?
A credible result shows cell-associated staining with interpretable nuclear or cytoplasmic localisation, assessed against a no-primary control (UniProt P06746: subcellular location; standard IHC practice). High staining in breast glandular cells provides a reference pattern, whereas adipocytes are reported as not detected (HPA: High in breast glandular cells; Not detected in adipocytes). Investigate signal confined to section edges, folds, necrotic regions, or sites of endogenous enzyme activity before calling those cells positive (standard IHC practice). Nuclear enrichment may fit the reported movement after DNA damage, but staining alone does not establish that damage occurred (UniProt P06746: subcellular location; standard IHC interpretation). Compare matched regions, controls, and processing batches before drawing that conclusion (standard IHC practice).
Boster reagents
Best POLB / DNA polymerase beta IHC Antibodies
The catalog includes an IHC image of paraffin-embedded rat lung for A01946-2 and lists human, mouse, rat, and zebrafish reactivity across the antibodies (A01946-2 image caption; catalog reactivity).
Real IHC data
Anti-DNA Polymerase Beta POLB Antibody
Cat # A01946-2
A01946-2 has an IHC image of paraffin-embedded rat lung with hematoxylin counterstain; its listed applications include IHC and ICC (A01946-2 image caption; catalog applications). M01946 lists IHC, IF, and ICC applications and human, mouse, and rat reactivity, but supplies no IHC or IF image (M01946 catalog applications, reactivity, and image fields).
Which to pick: Choose A01946-2 for tissue IHC based on its own paraffin-embedded rat lung image; the fixative is unreported (A01946-2 image caption). Choose M01946 for IF/ICC when a rabbit monoclonal is preferred; both applications are listed, but no IF image is supplied (M01946 catalog: host Rabbit, clone ACGH-16, applications IF and ICC, IF image field empty). For cross-species planning, A01946-2 lists human, mouse, rat, and zebrafish reactivity, while M01946 lists human, mouse, and rat; the sample-specific IHC evidence is limited to A01946-2’s rat lung image (catalog reactivity; A01946-2 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P06746 (DPOLB_HUMAN, DNA polymerase beta).
- Human Protein Atlas. POLB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. POLB subcellular location (ICC-IF): Localized to vesicles and cytosol..
- Human Protein Atlas. POLB antibody validation summary (4 antibodies).
- Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation. Frontiers in immunology 2022 — PMC9869049.
- Silencing DNA Polymerase β Induces Aneuploidy as a Biomarker of Poor Prognosis in Oral Squamous Cell Cancer. International journal of molecular sciences 2021 — PMC7957714.
- Integration of single-cell and bulk RNA sequencing reveals programmed cell death-associated transcriptional programs in sepsis-induced acute lung injury. PloS one 2026 — PMC13232957.
- A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer. Journal of Cancer 2016 — PMC4964131.
- PubMed PMID:10648175 — UniProt-cited evidence.
- PubMed PMID:7705833 — UniProt-cited evidence.
- PubMed PMID:7914364 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for POLB (DNA polymerase beta). Expected localisation General nuclear expression in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A01946-2 at 1:50-1:200 (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 rat lung tissue; fixative not specified (datasheet A01946-2); 2. Fixation: Image fixative and duration unreported (datasheet A01946-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-POLB, 1:50-1:200 (datasheet A01946-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01946-2); Caveat: DNA damage can shift POLB into nuclei (UniProt); Isoform / epitope: One 1–335 chain; no isoforms annotated (UniProt); Regulation: DNA damage changes nuclear localisation (UniProt). Evidence: UniProt P06746, HPA tissue IHC · Enhanced, HPA ICC-IF · vesicles and cytosol., Datasheet A01946-2, PMC9869049, PMC7957714. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/POLB/immunohistochemistry-ihc-guide.