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CCT3 / T-complex protein 1 subunit gamma · IHC design guide
Design Immunohistochemistry for CCT3
Plan chromogenic IHC on paraffin sections with the IHC-validated CCT3 antibody at 0.5–1 μg/mL (datasheet: PB9926). Use the mainly cytoplasmic lymphoid pattern and high staining in breast glandular cells versus low staining in esophageal squamous cells to plan controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCT3 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 | Mainly cytoplasmic (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in lymphoid tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9926) | |
| Positive control | Adrenal gland+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 | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | Two isoforms; isoform-specific epitope coverage is unknown (UniProt) |
Section 1
Recommended CCT3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9926) is accompanied by two published CCT3 tissue protocols (PMC13514722; PMC5958781).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet PB9926) |
| Fixation | Image fixative and duration unreported (datasheet PB9926); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9926) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9926) |
| Primary antibody | Rabbit anti-CCT3, 0.5-1μg/ml (datasheet PB9926) |
| Primary incubation | Overnight at 4 °C (datasheet PB9926) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9926) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCT3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic expression in lymphoid tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet: PB9926); both published protocols use heated citrate buffer (PMC13514722; PMC5958781).
Section 2
What Is the Expected CCT3 Staining Pattern?
CCT3 is a cytoplasmic chaperonin subunit with no transmembrane segment (UniProt P49368: subcellular location and topology). In paraffin sections, expect mainly cytoplasmic staining in lymphoid tissues and strong staining in selected glandular, glial, and squamous epithelial cells (HPA tissue IHC: profile and High observations). HPA rates the tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).
| Cytoplasmic staining in lymphoid tissue, or High staining in breast glandular cells. | This fits the reported tissue pattern and cytoplasmic location (HPA tissue IHC: profile; High in breast glandular cells; UniProt P49368: cytoplasm). Assess the named cell population rather than treating every cell in the section as an equivalent positive control. |
| A nuclear-only pattern, or a sharp membrane-only outline with little cytoplasmic signal. | These are poor matches for the expected dominant IHC pattern (HPA tissue IHC: mainly cytoplasmic; UniProt P49368: cytoplasm, no transmembrane segment). Review morphology and controls before interpreting them as CCT3. HPA does report an additional plasma membrane location in ICC-IF, so minor peripheral fluorescence alone is not grounds for rejection (HPA subcellular ICC-IF: plasma membrane). |
| Strong staining confined to a cell population outside the reported High populations. | Consider cross-reactivity or endogenous detection activity, then check a no-primary control. Do not label an unlisted population inherently negative: HPA reports low tissue RNA specificity and supplies no negative IHC cell list (HPA tissue IHC: RNA specificity; negative list). |
| Haze over cells and extracellular areas that obscures cell boundaries. | Treat diffuse background as an interpretation problem, especially if a no-primary control also develops color. Recheck the general IHC blocking, washing, and detection steps; the supplied sources do not identify a CCT3-specific cause of this appearance. |
| No convincing signal in breast glandular cells or cerebral cortex glial cells. | These are reported High populations (HPA tissue IHC: breast glandular cells; cerebral cortex glial cells). Check tissue integrity and the IHC workflow before concluding that CCT3 is absent. One failed section cannot overturn the reference pattern. |
💡Expected CCT3 appearanceCall a section positive when identifiable cells show predominantly cytoplasmic signal, with High staining plausible in breast glandular cells or cerebral cortex glial cells (UniProt P49368: cytoplasm; HPA tissue IHC: High in both); nuclear-only staining or diffuse color without cell boundaries is suspect.
| Dominant compartment | Interpret chromogenic signal against a cytoplasmic expectation (UniProt P49368: cytoplasm; HPA tissue IHC: mainly cytoplasmic). HPA's approved cytosol localization comes from ICC-IF, which offers compartment context but is a different application (HPA subcellular ICC-IF: cytosol approved). |
| Cell population and tissue | The expected intensity depends on the cells examined: HPA reports High staining in adrenal glandular cells and caudate glial cells, but Low staining in smooth muscle cells and ovarian stroma cells (HPA tissue IHC: listed levels). Low is not a validated negative control. |
| Strength of tissue evidence | HPA calls its tissue IHC reliability Approved while describing only medium consistency with RNA expression (HPA tissue IHC: reliability description). Use the reported pattern as a reference for review, not as proof that every specimen or cell must stain identically. |
| Antibody and application evidence | HPA006543 has Approved IHC and ICC status; HPA074177 has Approved ICC status with no listed IHC status (HPA antibodies: validation entries). An ICC result with the latter does not establish how it performs in paraffin IHC. |
| Protein forms and topology | UniProt lists 2 isoforms, a chain spanning residues 1–545, and no signal peptide, propeptide, or transmembrane segment (UniProt P49368: isoforms, processing, topology). These annotations do not identify the antibody epitope or predict antigen retrieval or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known High cell population is blank. | The result conflicts with the reference pattern if the sampled cells are breast glandular cells or caudate glial cells (HPA tissue IHC: High in both). A staining run or tissue-quality problem remains possible. | Confirm the intended cells are present and preserved, then check the catalog antibody's IHC-P instructions, retrieval step, dilution, detection reagents, and a known-positive section. Do not infer CCT3 loss from a single blank slide. |
| Only nuclei develop strong chromogen. | Nuclear-only signal disagrees with the dominant cytoplasmic expectation (UniProt P49368: cytoplasm; HPA tissue IHC: mainly cytoplasmic). Its source cannot be determined from location alone. | Compare the no-primary control and examine whether chromogen follows nuclei or overlaps cytoplasm. Review blocking and detection steps, then repeat with an independently stained positive section if the pattern persists. |
| Unexpected cells stain more strongly than the reported cell population. | Cross-reactivity or endogenous detection activity is possible. HPA has no negative IHC cell list, and its RNA specificity is Low (HPA tissue IHC: negative list and RNA specificity). | Check a no-primary control and compare cell morphology with a reference positive population. Report the actual stained cells; avoid declaring the unexpected population CCT3-positive solely from chromogen. |
| Color spreads through the section or appears in the no-primary control. | Background may arise from the general chromogenic detection workflow; neither UniProt nor HPA identifies a CCT3-specific background mechanism. | Review blocking, washing, and detection timing using standard IHC controls. Interpret target staining only where cell boundaries and the expected cytoplasmic compartment remain clear. |
| Smooth muscle or ovarian stroma stains weakly. | Weak signal may agree with the reported Low levels in these cell populations (HPA tissue IHC: smooth muscle cells; ovarian stroma cells). It does not, by itself, show that the run failed. | Judge the run alongside a reported High population and the no-primary control. Record cell-specific intensity rather than scoring an entire mixed tissue as uniformly positive or negative. |
| What should an IF/ICC image show? | HPA reports approved cytosol localization with additional approved vesicle and plasma membrane locations, based on ICC-IF (HPA subcellular ICC-IF: locations). This does not replace the paraffin IHC interpretation. | For an IF/ICC image, look for a dominant cytosolic signal and assess additional punctate or peripheral signal against controls (HPA subcellular ICC-IF: locations). Use the separate IF/ICC guide for that application's workflow. |
Experiment evidence
IHC & IF images showing CCT3 staining
PB9926 · IHCIHC analysis of CCT3 using anti-CCT3 antibody (PB9926). CCT3 was detected in paraffin-embedded section of human lung cancer tissue. Heat med
PB9926 · IHCIHC analysis of CCT3 using anti-CCT3 antibody (PB9926). CCT3 was detected in paraffin-embedded section of human mammary cancer tissue. Heat
PB9926 · IFIF analysis of CCT3 using anti-CCT3 antibody (PB9926). CCT3 was detected in immunocytochemical section of A431 cell. Enzyme antigen retrieva
M05920 · IFIF analysis of CCT3 using anti-CCT3 antibody (M05920). CCT3 was detected in immunocytochemical section of A431 cells. Enzyme antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163468-CCT3/tissue/cerebral+cortex#imid_2171204
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163468-CCT3/tissue/colon#imid_2171122
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163468-CCT3/tissue/liver#imid_2171131
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000163468-CCT3/tissue/kidney#imid_2171140
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006543; green = CCT3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006543; green = CCT3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCT3 IHC & IF
🧪Start with cerebral cortex; glial cells should stain strongly (HPA: High in cerebral-cortex glial cells). HPA detects CCT3 in all 45 scored tissues and lists no negative tissue (HPA: no negative rows), so use no-primary and isotype controls for negative comparisons; treat unstained adjacent cells as background references, not confirmed CCT3-negative cells.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CCT3 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 CCT3 in A-431, U-251MG, U2OS, K-562, OE19, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Run a no-primary (secondary-only) section, a rabbit IgG control matched to the primary antibody’s clonality and concentration, and a validated CCT3-knockout specimen if available (PB9926 caption: rabbit primary; standard IHC practice). Block endogenous peroxidase and biotin for the biotin-based DAB method, and check cerebral-cortex pigment against control sections before scoring brown signal (PB9926 caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9926 paraffin-section caption does not state a fixative (PB9926 caption). Heat retrieval in citrate buffer at pH 6 for 20 minutes is documented, but whether CCT3 detection depends on retrieval is unreported (PB9926 caption). Paraffin-section IHC is demonstrated in human lung cancer tissue, while the supplied ICC-IF images do not establish that IF or frozen sections are easier; cerebral-cortex lipofuscin can complicate DAB interpretation and autofluoresce in IF (PB9926 caption; HPA: ICC-IF images; standard histology).
HPA tissue IHC evidence for CCT3
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CCT3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CCT3 IHC Tips
Troubleshoot CCT3 staining in paraffin sections using the catalog antibody’s documented IHC conditions, then assess signal against expected cytoplasmic localisation.
What retrieval conditions should I try when CCT3 staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet PB9926; PB9926 tissue-IHC caption). The documented paraffin-section image used this treatment before incubation with 1 μg/ml primary antibody overnight at 4°C (PB9926 tissue-IHC caption). If staining remains weak, compare a longer heat exposure or a different retrieval buffer on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Excessive heating can damage tissue morphology or increase background, so inspect both signal and section integrity (standard IHC practice). Judge improvement by cytoplasmic staining in expected cells, with the same exposure and scoring rules across conditions (UniProt P49368 localisation; standard IHC practice).
Can fixation explain variable CCT3 staining between paraffin blocks?
Target-specific sensitivity of CCT3 to fixation is unknown from the supplied evidence; the PB9926 paraffin-section caption does not state a fixative (PB9926 tissue-IHC caption). Record each block’s fixative and fixation history before comparing staining, because fixation can alter antigen accessibility in routine IHC (standard IHC practice). Process matched sections together with the documented citrate pH 6, 20-minute retrieval and 1 μg/ml primary incubation to limit procedural variation (PB9926 tissue-IHC caption; standard IHC practice). If blocks differ in staining, assess morphology and a control section before assigning the difference to CCT3 abundance (standard IHC practice). Cytoplasmic localisation and annotated modifications do not establish a fixation response (UniProt P49368).
Where should convincing CCT3 signal appear in tissue sections?
Expect predominantly cytoplasmic staining: UniProt assigns CCT3 to the cytoplasm, and HPA describes mainly cytoplasmic expression in lymphoid tissues (UniProt P49368 localisation; HPA tissue IHC). HPA subcellular imaging places CCT3 mainly in the cytosol, with additional vesicle and plasma-membrane localisation (HPA subcellular, approved). Evaluate those patterns within intact cells using a counterstain that preserves cellular boundaries (HPA subcellular; standard IHC practice). CCT3 has no annotated transmembrane segment, so an exclusively sharp membranous outline warrants checking background and morphology (UniProt P49368 topology; standard IHC practice). Compare staining with a processed control section before treating an unexpected compartment as biological redistribution (standard IHC practice).
Could an epitope or isoform difference explain discordant CCT3 staining?
UniProt lists 2 CCT3 isoforms, but the supplied antibody evidence does not map the PB9926 epitope or establish isoform recognition (UniProt P49368 isoforms; PB9926 tissue-IHC caption). Do not interpret unequal staining as an isoform difference without an epitope map and an independently validated isoform-specific assay (standard IHC practice). Annotated CCT3 modifications include acetylation and phosphorylation, but their effect on this antibody’s tissue staining is unknown (UniProt P49368 modified residues). First compare matched sections using citrate pH 6 retrieval for 20 minutes and consistent detection (PB9926 tissue-IHC caption; standard IHC practice). Record any change in cytoplasmic signal alongside morphology and background before making a mechanistic claim (UniProt P49368 localisation; standard IHC practice).
How should I investigate CCT3 localisation by multiplex IF?
Treat IF as a separately optimised assay; the PB9926 caption documents chromogenic staining of paraffin sections, without IF fixation or permeabilisation conditions (PB9926 tissue-IHC caption). Multiplex CCT3 with a validated marker for the cell type being evaluated, and include single-channel controls to assess spectral bleed-through (standard IF practice). Choose a far-red CCT3 fluorophore if the tissue shows strong shorter-wavelength autofluorescence, then check an unstained section at the intended settings (standard IF practice). Because the principal expected pool is cytosolic and CCT3 has no transmembrane segment, optimise permeabilisation for intracellular access while monitoring cell boundaries (HPA subcellular; UniProt P49368 topology; standard IF practice). Assess cytosolic signal separately from HPA’s additional vesicle and plasma-membrane locations (HPA subcellular, approved).
What should I check when brown signal obscures CCT3 staining?
The documented PB9926 workflow used 10% goat serum, a biotinylated secondary antibody, a streptavidin–biotin complex and DAB (PB9926 tissue-IHC caption). Check a no-primary section for secondary or detection-system signal, and apply a peroxidase block when using DAB (standard IHC practice). If diffuse brown staining persists, assess endogenous biotin contribution to the biotin-based detection system and compare an appropriate alternative detection chemistry (PB9926 tissue-IHC caption; standard IHC practice). Keep development conditions consistent across test and control sections so differences remain interpretable (standard IHC practice). Prefer signal with intact cellular boundaries and the expected cytoplasmic pattern over pigment, precipitate or acellular staining (UniProt P49368 localisation; standard IHC practice).
How can I compare CCT3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then use cytoplasmic intensity and percentage of positive cells to calculate an H-score on a 0–300 scale (UniProt P49368 localisation; standard IHC practice). Alternatively, report percentage positive cells or positive-cell density per mm² when those measures better fit the specimen (standard IHC practice). Normalise each measure to the number of evaluable cells or viable tissue area, and exclude folds, necrosis and unstained edges by a consistent rule (standard IHC practice). Keep retrieval, antibody incubation, DAB development and image settings consistent between sections (PB9926 tissue-IHC caption; standard IHC practice). Report cell type and compartment with every score because CCT3 staining can occur across multiple tissue cell populations (HPA tissue IHC).
How do I distinguish true CCT3 staining from artefact?
Look for reproducible signal within intact cytoplasm; CCT3 is assigned to the cytoplasm, while HPA reports mainly cytosolic staining with additional vesicle and plasma-membrane localisation (UniProt P49368 localisation; HPA subcellular, approved). Check whether the stained cells fit the section: HPA reports high staining in adrenal and breast glandular cells and low staining in smooth-muscle cells (HPA tissue IHC). Discount isolated section-edge signal, necrotic areas and extracellular DAB deposits when judging cellular positivity (standard IHC practice). A no-primary control helps identify endogenous enzyme or detection-system staining in the chromogenic workflow (standard IHC practice). Interpret discordance cautiously because HPA rates tissue staining as Approved with medium consistency against RNA expression (HPA tissue IHC).
Boster reagents
Best CCT3 / T-complex protein 1 subunit gamma IHC Antibodies
Anti-CCT3 antibodies have IHC images from human paraffin sections (PB9926 image captions) and IF images from A431 cells (PB9926 and M05920 image captions); PB9926 lists human, mouse and rat reactivity (catalog).
Real IHC data
Anti-CCT3 Antibody ®
Cat # PB9926
Real IF data
Anti-CCT3 Antibody ® (monoclonal, 12H4)
Cat # M05920
PB9926 has IHC images from paraffin sections of human lung, mammary and intestinal cancers and placenta, plus IF images from A431 cells (PB9926 image captions). M05920 is listed for human IF/ICC and has an IF image from A431 cells (M05920 applications, reactivity and image caption).
Which to pick: Choose PB9926 for tissue IHC: its rabbit antibody is listed for IHC and shown on human paraffin sections; the captions do not report the fixative (PB9926 catalog and IHC image captions). For IF/ICC, M05920 is a mouse monoclonal clone 12H4 with a human A431 IF image; PB9926 also has A431 IF images (M05920 and PB9926 catalog and IF image captions). For mouse or rat samples, PB9926 is the listed cross-species option, but its paraffin IHC images and stated IHC dilution cover human samples only (PB9926 reactivity, dilution listing and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49368 (TCPG_HUMAN, T-complex protein 1 subunit gamma).
- Human Protein Atlas. CCT3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CCT3 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane and vesicles..
- Human Protein Atlas. CCT3 antibody validation summary (2 antibodies).
- Expression patterns and clinical implications of chaperonin subunit 3 mRNA and protein in laryngeal squamous cell carcinoma. World journal of clinical oncology 2025 — PMC12754133.
- CCT3 Enhances Lung Adenocarcinoma Progression by Promoting p53 Ubiquitination and Degradation. Medicina (Kaunas, Lithuania) 2026 — PMC13514722.
- Insights into the roles and driving forces of CCT3 in human tumors. Frontiers in pharmacology 2022 — PMC9596777.
- Suppression of CCT3 inhibits malignant proliferation of human papillary thyroid carcinoma cell. Oncology letters 2018 — PMC5958781.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCT3 (T-complex protein 1 subunit gamma). Expected localisation Mainly cytoplasmic (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9926). Validated IHC antibody: PB9926 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); 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 lung cancer tissue; fixative not specified (datasheet PB9926); 2. Fixation: Image fixative and duration unreported (datasheet PB9926); 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: Citrate pH 6 , 20 min (datasheet PB9926); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9926); 8. Primary antibody: Rabbit anti-CCT3, 0.5-1μg/ml (datasheet PB9926); 9. Primary incubation: Overnight at 4 °C (datasheet PB9926). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: Two isoforms; isoform-specific epitope coverage is unknown (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P49368, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet PB9926, PMC13514722, PMC5958781. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCT3/immunohistochemistry-ihc-guide.