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POLR3H / DNA-directed RNA polymerase III subunit RPC8 · IHC design guide
Design Immunohistochemistry for POLR3H
Plan POLR3H paraffin IHC with the catalog antibody at 1:100–1:300 (datasheet A30654). Compare the expected nuclear location (UniProt) with the observed nuclear and cytoplasmic tissue profile, using Sertoli cells as a high-staining reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
POLR3H 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 | Nuclear and cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | General nuclear and cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30654) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent between paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Medium IHC–RNA consistency; verify the pattern (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | Two isoforms; epitope impact unspecified (UniProt) |
Section 1
Recommended POLR3H IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published POLR3H protocol using formalin-fixed, paraffin-embedded breast cancer specimens (PMC13186826).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A30654) |
| Fixation | Image fixative and duration unreported (datasheet A30654); 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 8.0 (datasheet A30654); 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-POLR3H, 1:100 - 1:300 (datasheet A30654) |
| 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 | POLR3H-positive staining in sertoli cells of testis (HPA tissue IHC: High). HPA tissue profile: General nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA pH 8.0 retrieval (datasheet A30654); the published protocol reports antigen retrieval without specifying its method (PMC13186826).
Section 2
What Is the Expected POLR3H Staining Pattern?
POLR3H is a nuclear RNA polymerase III subunit with no transmembrane segment (UniProt Q9Y535). In paraffin sections, expect prominent staining in testicular Sertoli cells and moderate staining in several glandular, epithelial, hematopoietic, and neuronal cell types (HPA tissue IHC). HPA describes a general nuclear and cytoplasmic pattern, but rates its tissue IHC evidence Approved, with medium consistency against RNA expression and external verification pending (HPA tissue IHC).
| Strong Sertoli-cell staining with discernible nuclei in testis. | This matches the clearest listed tissue reference: Sertoli cells are High (HPA tissue IHC). Assess the cellular distribution alongside morphology; HPA reports general nuclear and cytoplasmic tissue staining, while UniProt assigns POLR3H to the nucleus (HPA tissue IHC; UniProt Q9Y535). |
| Moderate staining in glandular, respiratory epithelial, hematopoietic, or neuronal cells. | This can fit the reported pattern: adrenal, appendix, and breast glandular cells; bronchial respiratory epithelial cells; marrow hematopoietic cells; and cortical neurons are Medium (HPA tissue IHC). Identify the stained cells before comparing intensity across sections (general IHC practice). |
| Predominantly cell-surface or extracellular staining, without convincing cellular staining. | Treat this as a pattern requiring investigation: UniProt places POLR3H in the nucleus and lists no transmembrane segment, while HPA describes nuclear and cytoplasmic tissue staining (UniProt Q9Y535; HPA tissue IHC). Check section morphology and staining controls for artefact (general IHC practice). |
| Strong adipocyte staining or staining that obscures cell boundaries throughout the section. | Adipocytes are Not detected in the supplied HPA tissue IHC result (HPA tissue IHC). Check for cross-reactivity, endogenous detection activity, or nonspecific background before assigning the signal to POLR3H (general IHC practice). Other cell types require their own evidence because HPA reports low tissue specificity (HPA tissue IHC). |
| No staining in a technically adequate testis section containing Sertoli cells. | This conflicts with the listed High Sertoli-cell result (HPA tissue IHC). Review tissue identification, section quality, antibody and detection controls, and the general antigen-retrieval workflow before interpreting the sample as POLR3H-negative (general IHC practice). |
💡Expected POLR3H appearanceA convincing positive is High Sertoli-cell staining with a discernible nuclear component (HPA tissue IHC; UniProt Q9Y535); strong adipocyte or isolated surface staining warrants investigation (HPA tissue IHC; UniProt Q9Y535).
| Tissue and cell context | HPA reports Low tissue specificity by RNA and a general nuclear and cytoplasmic tissue IHC profile (HPA tissue IHC). Compare named cell populations: Sertoli cells are High, several listed populations are Medium, and adipocytes are Not detected (HPA tissue IHC). |
| Antibody validation | The listed HPA046787 antibody is Approved for IHC; its tissue profile has medium consistency with RNA expression and awaits external verification (HPA antibodies; HPA tissue IHC). Use controls when a sample differs from the reference pattern (general IHC practice). |
| Isoforms and epitope coverage | UniProt lists 2 POLR3H isoforms, but the supplied evidence gives no antibody epitope or isoform-specific staining result (UniProt Q9Y535; HPA antibodies). Avoid assigning a staining difference to a particular isoform without separate validation (general IHC practice). |
| What should IF/ICC show? | HPA reports mainly nucleoplasmic localization, with additional vesicle and centrosome signals; its listed antibody is Supported for ICC (HPA subcellular; HPA antibodies). Those cell-image observations provide context for compartment assessment but do not establish an IHC retrieval condition (HPA subcellular; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Sertoli cells show little or no signal. | The expected High reference signal is absent; the cause cannot be assigned from the supplied tissue data alone (HPA tissue IHC). | Confirm Sertoli cells are present, then check positive-control staining, antibody handling, retrieval, and detection steps (general IHC practice). |
| Most cells and extracellular spaces have diffuse chromogen. | Nonspecific background or detection activity can obscure cellular localization (general IHC practice). | Inspect a no-primary control and review blocking, washes, and chromogen development; reassess identifiable cells afterward (general IHC practice). |
| Adipocytes stain strongly. | This differs from the Not detected adipocyte result (HPA tissue IHC); cross-reactivity or background is possible (general IHC practice). | Compare with a no-primary control and a Sertoli-cell positive control before calling adipocyte signal specific (general IHC practice; HPA tissue IHC). |
| Signal appears mainly on cell surfaces. | A dominant surface pattern does not match the reported nuclear and cytoplasmic profile or the absence of a transmembrane segment (HPA tissue IHC; UniProt Q9Y535). | Check morphology, section edges, and detection controls; score convincing intracellular staining separately (general IHC practice). |
| The nuclear component is difficult to judge. | Dense chromogen or an unclear counterstain can hide compartment boundaries (general IHC practice). | Review development and counterstaining, then compare nuclei and cytoplasm in intact cells (general IHC practice; HPA tissue IHC). |
| A weakly stained tissue differs from a reference image. | The HPA IHC profile has medium consistency with RNA expression and awaits external verification (HPA tissue IHC). | Confirm the same cell type is being compared and interpret the difference with section controls; avoid inferring a POLR3H-specific fixation effect from these data (general IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing POLR3H staining
A30654 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
A13065-1 · IHCImmunohistochemistry validation of POLR3H using Anti-POLR3H Antibody (A13065-1). Immunohistochemical analysis of paraffin-embedded Human bra
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100413-POLR3H/tissue/cerebral+cortex#imid_13218351
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100413-POLR3H/tissue/colon#imid_13212813
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100413-POLR3H/tissue/liver#imid_13212806
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100413-POLR3H/tissue/kidney#imid_13212815
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA046787; green = POLR3H, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA046787; green = POLR3H, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for POLR3H IHC & IF
🧪Run testis first: Sertoli cells should stain (HPA: High in Sertoli cells), and use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes). On the testis slide, compare Sertoli-cell nuclei with adjacent cells for background, but do not designate those cells as true negatives because HPA provides no internal negative cell type (HPA: testis Sertoli cells High).
Positive control tissue: Testis (Sertoli cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show POLR3H in A-431, HEK293, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and primary-matched isotype controls, matching the primary antibody’s host species and clonality where applicable; use POLR3H knockout material or an immunogen-peptide block as a biological specificity control (selected-SKU IHC caption: peptide-block negative control). Block endogenous peroxidase for chromogenic testis IHC, and check endogenous biotin background if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU IHC caption: fixative unreported). That brain IHC example uses high-pressure, high-temperature Tris–EDTA retrieval at pH 8.0 and antibody at 1:100 overnight at 4 °C; it does not establish that retrieval is required for testis (selected-SKU IHC caption). Frozen sections or IF are not established as easier by the supplied evidence; for testis chromogenic IHC, assess endogenous peroxidase background against the controls (HPA: testis Sertoli cells High; standard IHC practice).
HPA tissue IHC evidence for POLR3H
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Testis | Sertoli cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | 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 → |
Section 3
Advanced POLR3H IHC Tips
Troubleshoot POLR3H staining in paraffin sections by checking retrieval, nuclear localisation, controls and cell-specific scoring before interpreting differences between specimens.
How should I adjust retrieval when POLR3H staining is weak in paraffin sections?
Start with heat-mediated Tris-EDTA retrieval at pH 8.0 (datasheet A30654). The selected paraffin-section image used high-pressure, high-temperature retrieval in that buffer, followed by antibody at 1:100 overnight at 4°C (caption A30654). If nuclear staining remains weak, compare a modestly longer retrieval with the original conditions on adjacent sections, keeping detection and imaging settings constant (general IHC practice). Inspect tissue integrity and background alongside signal: excessive heating can damage sections, so a brighter stain alone does not establish better retrieval (general IHC practice).
Could fixation explain weak or uneven POLR3H staining?
POLR3H-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A30654). Record the specimen’s actual fixation and processing history before comparing staining across blocks, since fixation can change antigen accessibility in IHC (general IHC practice). Compare sections with similar processing, thickness, retrieval and detection settings, and check whether uneven staining follows folds, damaged areas or section edges (general IHC practice). Keep the catalog antibody’s pH 8.0 Tris-EDTA retrieval as the starting condition while assessing those variables (datasheet A30654).
Should cytoplasmic POLR3H staining be considered specific?
Expect a nuclear component because UniProt places POLR3H in the nucleus and identifies it as part of the RNA polymerase III stalk (UniProt Q9Y535). HPA tissue IHC reports general nuclear and cytoplasmic staining, so cytoplasmic colour alone cannot establish either specificity or an error (HPA tissue IHC). Evaluate nuclear and cytoplasmic staining separately in intact cells, using the counterstain to define nuclei and adjacent sections to judge reproducibility (general IHC practice). Give greater weight to the expected nuclear pattern and to loss of staining in a matched peptide-absorption control; the selected image includes such a control (UniProt Q9Y535; caption A30654).
Can an unknown antibody epitope affect interpretation of POLR3H staining?
POLR3H has 2 annotated isoforms, but the supplied antibody caption does not map its epitope to either one (UniProt Q9Y535; caption A30654). Consequently, do not assign staining in paraffin sections to a particular isoform without epitope information or an isoform-selective control (general IHC practice). UniProt annotates no transmembrane segment, glycosylation sites or modified residues for the 204-amino-acid chain; these annotations do not establish how fixation exposes this antibody’s epitope (UniProt Q9Y535). If staining changes across processing conditions, compare the same tissue and cell population under matched detection settings before attributing the change to isoform expression (general IHC practice).
How can I cross-check POLR3H staining by multiplex IF?
On the separate IF/ICC workflow, pair POLR3H with a validated marker for the expected cell type; Sertoli cells are an HPA high-staining population (HPA tissue IHC). Choose spectrally separated fluorophores and favour a red or far-red POLR3H channel if tissue autofluorescence obscures shorter wavelengths (general IF practice). Because POLR3H has no transmembrane segment and its main supported IF location is nucleoplasmic, permeabilise fixed cells sufficiently for antibody access through the plasma and nuclear envelopes (UniProt Q9Y535; HPA subcellular). Compare nuclear signal with the counterstain and include single-channel controls to distinguish overlap from bleed-through or autofluorescence (general IF practice).
How do I distinguish POLR3H staining from chromogenic background?
First compare intact nuclear staining with blank regions, damaged tissue and the peptide-absorption control shown for the selected paraffin-section image (UniProt Q9Y535; caption A30654). Include a no-primary control to assess secondary-reagent or detection background, and apply a peroxidase block before chromogenic detection when using a peroxidase-based workflow (general IHC practice). Review diffuse DAB deposits, pigment and strong edge staining separately from cell-associated signal, using the counterstain to locate nuclei (general IHC practice). If background dominates, adjust blocking, washing or detection conditions one at a time while retaining the documented 1:100 antibody condition as a comparison (caption A30654; general IHC practice).
What is a defensible way to quantify POLR3H IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring compartment before measuring POLR3H; nuclear scoring aligns with its UniProt location, while HPA also reports cytoplasmic tissue staining (UniProt Q9Y535; HPA tissue IHC). For chromogenic sections, report the percentage of positive intact cells and, if intensity is reproducibly graded, an H-score on the standard 0–300 scale (general IHC practice). Alternatively, report positive-cell density per mm² of viable tissue, specifying the area examined and excluding folds or necrosis (general IHC practice). Normalise comparisons to the same cell population, viable area, staining run and detection settings; keep nuclear and cytoplasmic results separate (general IHC practice).
When should a POLR3H-positive area be treated cautiously?
A credible result includes cell-associated nuclear staining consistent with POLR3H’s annotated nuclear location, assessed against the section’s counterstain (UniProt Q9Y535; general IHC practice). HPA reports high staining in Sertoli cells and no detected staining in adipocytes, but its tissue IHC reliability remains pending external verification (HPA tissue IHC). Treat colour limited to the wrong compartment or unexpected cells cautiously, particularly if it follows edges, necrosis or endogenous peroxidase activity (general IHC practice). Check adjacent sections and no-primary controls, and compare with the selected image’s peptide-absorption control before assigning biological meaning to a disputed pattern (caption A30654; general IHC practice).
Boster reagents
Best POLR3H / DNA-directed RNA polymerase III subunit RPC8 IHC Antibodies
Both antibodies have IHC images of paraffin-embedded human brain (catalog IHC captions); both list IF and human, mouse, and rat reactivity (catalog applications/reactivity).
Real IHC data
Anti-RPC8 Antibody
Cat # A30654
Real IHC data
Anti-POLR3H Antibody
Cat # A13065-1
A30654 has an IHC image of paraffin-embedded human brain with retrieval and peptide pre-absorption control details (A30654 IHC caption). A13065-1 has an IHC image of paraffin-embedded human brain and lists IF as an application (A13065-1 IHC caption; catalog applications).
Which to pick: For tissue IHC, start with A30654: its paraffin-section caption reports retrieval and a peptide pre-absorption control; the fixative is unreported (A30654 IHC caption). For IF/ICC, choose A30654 if ICC is needed, since it lists both applications; A13065-1 lists IF (catalog applications). Both list human, mouse, and rat reactivity, but their IHC images show human brain only (catalog reactivity; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y535 (RPC8_HUMAN, DNA-directed RNA polymerase III subunit RPC8).
- Human Protein Atlas. POLR3H tissue IHC expression (reliability: Approved).
- Human Protein Atlas. POLR3H subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles and centrosome..
- Human Protein Atlas. POLR3H antibody validation summary (1 antibodies).
- Integrated Analysis of Bulk and Single-Cell Transcriptomics Identifies Prognostic Biomarkers in Breast Cancer Metastasis. Biochemical genetics 2026 — PMC13186826.
- PubMed PMID:12391170 — UniProt-cited evidence.
- PubMed PMID:11258795 — UniProt-cited evidence.
- PubMed PMID:15461802 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for POLR3H (DNA-directed RNA polymerase III subunit RPC8). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30654). Validated IHC antibody: A30654 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Sertoli cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human brain tissue; fixative not specified (datasheet A30654); 2. Fixation: Image fixative and duration unreported (datasheet A30654); 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 8.0 (datasheet A30654); 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-POLR3H, 1:100 - 1:300 (datasheet A30654); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent between paraffin sections. (standard IHC practice; not target-specific); Caveat: Medium IHC–RNA consistency; verify the pattern (HPA tissue IHC); Isoform / epitope: Two isoforms; epitope impact unspecified (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q9Y535, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A30654, PMC13186826. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/POLR3H/immunohistochemistry-ihc-guide.