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PSMC4 / 26S proteasome regulatory subunit 6B · IHC design guide
Design Immunohistochemistry for PSMC4
Plan chromogenic IHC for PSMC4 in paraffin sections using the catalog antibody’s documented workflow (datasheet A06099-1). Compare cytoplasmic and nuclear staining with the tissue profile, while allowing for medium consistency between staining and RNA data (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PSMC4 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 nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear signal across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06099-1) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended PSMC4 IHC & IF Protocols
The catalog antibody’s IHC protocol is accompanied by 2 published PSMC4 tissue staining protocols (PMC12081262; PMC10424298).
| Sample | Paraffin-embedded human glioblastoma tissue; fixative not specified (datasheet A06099-1) |
| Fixation | Image fixative and duration unreported (datasheet A06099-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A06099-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06099-1) |
| Primary antibody | Rabbit anti-PSMC4, 2-5 μg/ml (datasheet A06099-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06099-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06099-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PSMC4-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and nuclear expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A06099-1); the published excerpts do not specify retrieval conditions (PMC12081262; PMC10424298).
Section 2
What Is the Expected PSMC4 Staining Pattern?
PSMC4 should show cytoplasmic and nuclear staining across tissues, including glandular, neuronal and adipose cells represented in HPA images (HPA tissue IHC). It is a proteasome subunit with no transmembrane segment, consistent with an intracellular pattern (UniProt P43686). HPA rates the tissue IHC profile Approved, with medium consistency between antibody staining and RNA expression data (HPA tissue IHC).
| Cytoplasmic and nuclear signal appears in several cell populations, with moderate staining in a reference tissue. | This fits HPA's broad tissue profile. Adipocytes in adipose tissue, neuronal cells in cerebral cortex and glandular cells in adrenal gland are listed at Medium; compare the same cell type when judging intensity (HPA tissue IHC). |
| A sharply membrane-restricted pattern dominates while cytoplasm and nuclei remain clear. | That pattern conflicts with the reported cytoplasmic and nuclear distribution and the absence of a transmembrane segment. Check whether edge staining persists in the controls before interpreting it as PSMC4 (HPA tissue IHC; UniProt P43686 topology; general IHC practice). |
| Signal is confined to an unexpected cell population while the annotated cells in the same section are clear. | Review cell identification and controls; cross-reactivity or endogenous detection activity is possible in chromogenic IHC. Broad PSMC4 expression means an unlisted positive cell alone does not prove an artefact (HPA tissue IHC; general IHC practice). |
| Weak, diffuse colour covers tissue and background, obscuring cell boundaries. | The section cannot reliably establish cytoplasmic or nuclear localisation. Inspect the negative control and detection conditions before scoring; widespread colour alone is insufficient because HPA reports expression across tissues (HPA tissue IHC; general IHC practice). |
| No signal appears in the annotated cells of an HPA Medium tissue. | Treat this as a failed or inconclusive staining run until controls and tissue identity are checked. HPA lists Medium staining in several cell populations, but its Approved rating includes only medium consistency with RNA data (HPA tissue IHC). |
💡Expected PSMC4 appearanceCall a result positive when identifiable cells show cytoplasmic and nuclear staining, with Medium signal in an HPA Medium reference population; membrane-only colour or colour without cellular localisation is suspect (HPA tissue IHC; UniProt P43686 topology; general IHC practice).
| Subcellular distribution | UniProt places PSMC4 in cytoplasm and nucleus; HPA tissue IHC reports both compartments across tissues. Score both compartments rather than requiring one to dominate (UniProt P43686; HPA tissue IHC). |
| Choice of reference tissue and cells | HPA lists Medium staining for selected adipocytes, neuronal cells and glandular cells, and Low staining for thyroid glandular cells and lung alveolar cells. Low is not a negative control; no negative tissue is supplied (HPA tissue IHC). |
| Antibody evidence | HPA lists 2 antibodies with Approved IHC status, HPA002044 and HPA005471. The overall tissue profile has medium consistency with RNA data; agreement with the reported pattern supports interpretation without making every cell-level result certain (HPA antibodies; HPA tissue IHC). |
| Isoforms and processing | UniProt lists 2 isoforms, no signal peptide or propeptide, and a chain spanning residues 1–418. The payload gives no antibody epitope, so isoform coverage and epitope-dependent differences cannot be predicted (UniProt P43686). |
| IF/ICC cross-check | What pattern should IF/ICC show? Mainly nucleoplasm and cytosol; HPA also reports vesicles and other additional locations with differing confidence. This is a localisation cross-check, not an IHC-P protocol option (HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Annotated Medium reference cells are unstained. | The run may have failed, or tissue identification or detection may be wrong; the section alone cannot identify which (HPA tissue IHC; general IHC practice). | Verify tissue and cell identity, inspect the run control, then review the antibody and detection steps before assigning a biological negative (general IHC practice). |
| Only the nucleus or only the cytoplasm stains strongly. | Both compartments are expected, but their relative intensity is not specified for each cell type (HPA tissue IHC; UniProt P43686). | Compare matched cells with HPA's compartment pattern and check controls; avoid rejecting a run solely because one expected compartment is fainter (HPA tissue IHC; general IHC practice). |
| Membrane-like outlines dominate. | A membrane-restricted result is discordant with the reported intracellular distribution and no transmembrane segment (HPA tissue IHC; UniProt P43686 topology). | Review the counterstain and negative control, then reassess whether the apparent outlines contain cytoplasmic signal (general IHC practice). |
| Unexpected cells stain while annotated cells do not. | Misidentified cells, antibody cross-reactivity or endogenous chromogenic activity could explain the mismatch (HPA tissue IHC; general IHC practice). | Confirm morphology and compare detection controls; use HPA's named cell populations as references without treating other cell types as established negatives (HPA tissue IHC; general IHC practice). |
| Diffuse colour prevents compartment scoring. | Background from blocking, detection or wash conditions can obscure cellular signal; the payload does not identify a PSMC4-specific cause (general IHC practice). | Inspect negative controls, then adjust routine blocking, washing or detection conditions and rescore only when nuclei and cytoplasm are distinguishable (general IHC practice). |
| A Low HPA tissue gives a weak result. | HPA labels the listed cell population Low, so weak staining is compatible with the reference; Low does not mean absent (HPA tissue IHC). | Check a listed Medium population in the same run before calling the weak section a failed stain (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing PSMC4 staining
A06099-1 · IHCIHC analysis of PSMC4 using anti-PSMC4 antibody (A06099-1). PSMC4 was detected in a paraffin-embedded section of human glioblastoma tissue.
A06099-1 · IHCIHC analysis of PSMC4 using anti-PSMC4 antibody (A06099-1). PSMC4 was detected in a paraffin-embedded section of human lung adenocarcinoma t
A06099-1 · IFIF analysis of PSMC4 using anti-PSMC4 antibody (A06099-1) and anti-Beta Tubulin antibody (M01857-3). PSMC4 was detected in immunocytochemica
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000013275-PSMC4/tissue/cerebral+cortex#imid_1853592
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000013275-PSMC4/tissue/cerebellum#imid_1853603
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000013275-PSMC4/tissue/colon#imid_1853534
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000013275-PSMC4/tissue/liver#imid_1853519
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002044; green = PSMC4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002044; green = PSMC4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PSMC4 IHC & IF
🧪Run adipose tissue first and confirm staining in adipocytes, which HPA scores at Medium (HPA: adipose tissue, adipocytes, Medium). HPA detects PSMC4 in all 45 scored tissues, so there is no supported negative tissue; no cell population on the positive slide is established as PSMC4-negative, and any intact unstained cells should be treated as provisional internal comparators while no-primary and isotype controls establish background (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: None in HPA: PSMC4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PSMC4 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, NIH 3T3, with annotated localisation: Nucleoplasm (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only section and a rabbit IgG control matched to the primary antibody’s clonality and concentration where known; use PSMC4-depleted cells processed alongside the tissue as a biological negative (caption: rabbit anti-PSMC4; standard IHC practice). Quench endogenous peroxidase for DAB detection and assess nonspecific staining around adipose lipid spaces (caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06099-1 paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). Paraffin IHC has a documented starting point using heat retrieval in EDTA at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C; retrieval dependency has not been established by a comparison without retrieval (selected-SKU caption). Frozen sections or IF cannot be judged easier from the supplied evidence, although HPA ICC-IF supports nucleoplasmic and cytosolic localization; in adipose sections, distinguish staining of thin cellular rims from empty lipid spaces (HPA: supported nucleoplasm and cytosol; standard histology practice).
HPA tissue IHC evidence for PSMC4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: PSMC4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced PSMC4 IHC Tips
Troubleshoot PSMC4 staining in paraffin sections by checking retrieval, intracellular localisation, controls, and scoring before interpreting biological differences.
What should I change when PSMC4 staining is weak after antigen retrieval?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A06099-1). The catalog antibody stained a paraffin section of human glioblastoma after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A06099-1). If staining remains weak, compare retrieval heating and cooling conditions across matched sections while holding antibody concentration and detection constant (general IHC practice). Check whether the expected cytoplasmic and nuclear signal improves together, since both compartments are reported for PSMC4 (HPA tissue IHC; UniProt P43686). Include a section processed without primary antibody to distinguish recovered signal from detection background (general IHC practice).
How can I assess whether fixation is causing weak PSMC4 staining?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet A06099-1). Record the fixative, fixation interval, tissue thickness, and processing history for each section before comparing staining (general IHC practice). On matched material, hold EDTA retrieval at pH 8.0 and the primary concentration at 2 μg/ml while evaluating processing differences (datasheet A06099-1; general IHC practice). Examine preserved morphology and the balance of nuclear and cytoplasmic staining; PSMC4 is reported in both compartments (HPA tissue IHC; UniProt P43686). A processing association alone does not establish a PSMC4-specific fixation effect (general IHC practice).
Should PSMC4 appear in nuclei, cytoplasm, or both in tissue sections?
Expect cytoplasmic and nuclear staining in tissue sections, with nucleoplasm and cytosol supported by subcellular imaging (HPA tissue IHC; HPA subcellular). PSMC4 has no annotated transmembrane segment, so an exclusively membranous pattern warrants scrutiny (UniProt P43686 topology). Compare nuclear signal with a counterstain and assess cytoplasmic signal within intact cell boundaries (general IHC practice). Review the pattern across matched sections retrieved in EDTA at pH 8.0, rather than judging isolated dark cells (datasheet A06099-1; general IHC practice). If staining is confined to tissue edges or disrupted regions, inspect morphology and the no-primary control before assigning localisation (general IHC practice).
Can this IHC stain distinguish PSMC4 isoforms or modification states?
Two PSMC4 isoforms are listed, but the supplied evidence does not map this antibody’s epitope to either isoform (UniProt P43686; datasheet A06099-1). The record also lists phosphorylation and acetylation sites, including serines 21 and 28 and lysine 397 (UniProt P43686 modified residues). Therefore, treat chromogenic intensity as antibody-reactive PSMC4 staining, without assigning an isoform or modification state from this assay alone (general IHC practice). If a specific species matters, obtain an epitope map and validate specificity with an appropriate orthogonal control (general IHC practice). Keep EDTA retrieval at pH 8.0 consistent during that comparison (datasheet A06099-1).
How should I adapt the localisation check to multiplex IF?
For a separate IF/ICC experiment, pair PSMC4 with a marker for the cell population under study; HPA reports medium staining in neuronal cells of the cerebral cortex (HPA tissue IHC). Choose a fluorophore channel after measuring tissue autofluorescence and include single-stain controls to assess bleed-through (general IF practice). PSMC4 is reported in nucleoplasm and cytosol and has no annotated transmembrane segment, so permeabilisation should give antibody access to intracellular epitopes (HPA subcellular; UniProt P43686 topology; general IF practice). Optimise permeabilisation against preserved morphology and nuclear counterstaining rather than assuming the antibody epitope is mapped (general IF practice). Do not transfer the paraffin-section retrieval or 2 μg/ml concentration directly to IF without validation (datasheet A06099-1; general IF practice).
How do I troubleshoot diffuse brown background in PSMC4 IHC?
First compare the stained section with a no-primary control and inspect whether diffuse brown signal persists without the catalog antibody (general IHC practice). The demonstrated workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase-based DAB detection (datasheet A06099-1). Check the effectiveness of a peroxidase block and reagent-only controls because endogenous enzyme activity can contribute colour in chromogenic IHC (general IHC practice). Then assess washing, antibody concentration, and DAB development while keeping EDTA retrieval at pH 8.0 constant (datasheet A06099-1; general IHC practice). Retain nuclear and cytoplasmic signal as the localisation reference (HPA tissue IHC).
How should I score PSMC4 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear and cytoplasmic compartments separately because both are reported for PSMC4 (HPA tissue IHC; UniProt P43686). An H-score combines the percentage of cells at intensity grades 0–3, yielding 0–300; alternatively, report percentage positive cells or positive-cell density per mm² (general IHC practice). Normalise counts to viable cells or viable tissue area within the same annotated compartment (general IHC practice). Use the same staining batch, imaging settings, and positivity threshold across compared sections (general IHC practice). Record retrieval in EDTA at pH 8.0 and exclude necrotic and edge regions before scoring (datasheet A06099-1; general IHC practice).
When is PSMC4 staining convincing rather than artefactual?
A plausible positive pattern includes cytoplasmic and nuclear signal in intact cells, consistent with the reported tissue and subcellular localisation (HPA tissue IHC; HPA subcellular). Check that the stained cells match the annotated tissue population; HPA reports medium staining in adipocytes and in neuronal cells of the cerebral cortex (HPA tissue IHC). Question isolated membrane-only staining because PSMC4 lacks an annotated transmembrane segment (UniProt P43686 topology). Exclude staining concentrated at section edges or necrotic areas, and investigate signal in the no-primary control as possible detection background (general IHC practice). Interpret differences cautiously: HPA assigns its tissue staining Approved reliability with medium consistency against RNA expression (HPA tissue IHC).
Boster reagents
Best PSMC4 / 26S proteasome regulatory subunit 6B IHC Antibodies
A06099-1 has paraffin-section IHC images from human tumors and an IF image from human U2OS cells (catalog image captions). Both SKUs list human, mouse, and rat reactivity (catalog applications/reactivity).
Real IHC data
Anti-PSMC4 Antibody ®
Cat # A06099-1
A06099-1 has IHC images from paraffin-embedded human glioblastoma, lung adenocarcinoma, rectum adenocarcinoma, and urothelial carcinoma sections, plus an IF image from human U2OS cells (catalog image captions). M06099-1 lists IF/ICC applications and human, mouse, and rat reactivity, with no IHC or IF image supplied (catalog applications/reactivity; catalog image captions).
Which to pick: Choose A06099-1 for paraffin-section IHC: its human tissue captions document EDTA pH 8.0 retrieval and 2 μg/ml primary antibody, while the catalog lists an IHC range of 2–5 μg/ml for human and mouse (A06099-1 IHC image captions; catalog dilution). For IF/ICC, A06099-1 has a human U2OS IF image at 5 μg/ml; M06099-1 is a rabbit monoclonal, clone 31P63, listed for IF/ICC at 1:50–1:200 without an image (A06099-1 IF image caption; M06099-1 catalog applications/dilution). Both list human, mouse, and rat reactivity, but A06099-1's IHC dilution is specified for human and mouse only; the fixative used for its paraffin sections is unreported (catalog reactivity/dilution; A06099-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P43686 (PRS6B_HUMAN, 26S proteasome regulatory subunit 6B).
- Human Protein Atlas. PSMC4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PSMC4 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to vesicles, basal body, mid piece and principal piece..
- Human Protein Atlas. PSMC4 antibody validation summary (2 antibodies).
- The role of PSMC4 in non-small cell lung cancer: implications for prognosis, diagnosis, and immune microenvironment modulation. Frontiers in oncology 2025 — PMC12081262.
- Identification of an immune gene signature for predicting the prognosis of patients with uterine corpus endometrial carcinoma. Cancer cell international 2020 — PMC7650210.
- PSMC4 promotes prostate carcinoma progression by regulating the CBX3-EGFR-PI3K-AKT-mTOR pathway. Journal of cellular and molecular medicine 2023 — PMC10424298.
- PubMed PMID:8419915 — UniProt-cited evidence.
- PubMed PMID:11042152 — UniProt-cited evidence.
- PubMed PMID:8603043 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PSMC4 (26S proteasome regulatory subunit 6B). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06099-1). Validated IHC antibody: A06099-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human glioblastoma tissue; fixative not specified (datasheet A06099-1); 2. Fixation: Image fixative and duration unreported (datasheet A06099-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A06099-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06099-1); 8. Primary antibody: Rabbit anti-PSMC4, 2-5 μg/ml (datasheet A06099-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06099-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P43686, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A06099-1, PMC12081262, PMC10424298. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PSMC4/immunohistochemistry-ihc-guide.