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TSG101 / Tumor susceptibility gene 101 protein · IHC design guide
Design Immunohistochemistry for TSG101
Plan chromogenic TSG101 IHC around the cytoplasmic and membranous tissue pattern (HPA tissue IHC). This guide identifies high-staining cell populations for controls and flags cell-cycle-dependent nuclear localisation when interpreting unexpected staining (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TSG101 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 membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | High glandular staining; generally cytoplasmic and membranous (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Cell-cycle-dependent nuclear localisation may alter the pattern (UniProt) | |
| Regulation | Intensity regulation not specified (UniProt) | |
| Isoform / epitope | 2 isoforms; confirm antibody epitope coverage (UniProt) |
Section 1
Recommended TSG101 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published cervical and renal tissue IHC procedures (PMC3997686; PMC6332891).
| Sample | Paraffin-embedded Rat liver tissue; fixative not specified (datasheet M01233) |
| Fixation | Image fixative and duration unreported (datasheet M01233); 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 monoclonal (clone IDG-20) anti-TSG101, 1:50 recommended; image 1:500 (datasheet M01233) |
| 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 | TSG101-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and membranous expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval setting).
Section 2
What Is the Expected TSG101 Staining Pattern?
TSG101 is mainly cytoplasmic, with endosomal membrane association and cell cycle-dependent nuclear localisation (UniProt Q99816). In paraffin-section IHC, expect cytoplasmic and membranous staining in cell types such as appendix glandular cells, bone marrow hematopoietic cells and cerebral cortex neurons (HPA: High). HPA rates its tissue IHC evidence Approved, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Cytoplasmic staining, sometimes with a membranous component, is clear in glandular cells of appendix or colon (HPA: High in both). | This fits the reported tissue pattern and TSG101’s mainly cytoplasmic localisation (HPA: general cytoplasmic and membranous expression; UniProt Q99816). Compare cell compartments within the section; a positive call rests on cellular staining in the expected cells, not on colour anywhere in the tissue (general IHC practice). |
| Staining is exclusively extracellular or fills gland lumens, with little signal inside the expected cells. | That distribution does not fit the reported cellular pattern (HPA: general cytoplasmic and membranous expression). Treat it as suspect and inspect the section for nonspecific deposit or detection background before scoring TSG101 (general IHC practice). Nuclear staining alone needs separate judgment: UniProt reports cell cycle-dependent nuclear localisation (UniProt Q99816). |
| Strong colour appears chiefly in adipocytes while the expected positive cells are weak or unstained. | Adipocytes are reported as Not detected, so that reversal raises concern for nonspecific binding, pigment or endogenous detection activity (HPA: adipocytes Not detected; general IHC practice). It does not establish which cause is responsible. Compare a known-positive tissue and a primary-antibody omission control processed with the same detection system (general IHC practice). |
| A diffuse haze covers stroma, empty spaces and many cell types without a readable cellular pattern. | Broad haze cannot establish TSG101 localisation or identify positive cells (general IHC practice). Review blocking, washes, antibody concentration and detection background using matched controls (general IHC practice). HPA’s Approved IHC rating describes the reported staining evidence; it does not validate diffuse signal in a new run (HPA: IHC Approved). |
| No cellular signal appears in appendix glandular cells or bone marrow hematopoietic cells. | Both are reported High, making them useful positive comparators, although the supplied evidence does not guarantee every section or run will stain (HPA: High in appendix glandular cells and bone marrow hematopoietic cells). Check tissue preservation and the general IHC workflow, including retrieval, reagent activity and detection controls, before interpreting an unknown specimen as negative (general IHC practice). |
💡Expected TSG101 appearanceCall a positive IHC result when cytoplasmic, possibly membranous, staining is discernible in expected cells such as colon glandular cells or cerebral cortex neurons, both reported High; diffuse acellular colour alone is suspect (HPA: general pattern and High in both tissues; general IHC practice).
| Cell and tissue context | HPA reports High staining in several epithelial, hematopoietic and neuronal populations, but Not detected in adipocytes; judge intensity by cell type, not whole-section colour (HPA: tissue IHC; general IHC practice). |
| Membrane association | TSG101 associates with early and late endosome membranes and is mainly cytoplasmic; it has no transmembrane segment (UniProt Q99816). A membranous component can fit the HPA IHC profile without requiring an exclusively surface pattern (HPA: tissue IHC). |
| Cell division and nuclear signal | Nuclear localisation depends on cell cycle, and CEP55 interaction is required for midbody localisation during cytokinesis (UniProt Q99816). Assess a focal nuclear or division-associated signal in cellular context rather than assuming every such signal is artefact (general IHC practice). |
| IHC evidence strength | HPA lists HPA006161 and CAB004283 as IHC Approved, while its tissue profile has medium consistency with RNA expression (HPA: antibody validation; HPA: tissue IHC). These ratings support pattern comparison but do not establish the specificity of an individual slide. |
| Antigen retrieval | No target-specific retrieval condition or fixation sensitivity is supplied (UniProt Q99816; HPA: supplied records). If signal is weak, check the antibody’s validated IHC-P instructions and run controls when adjusting retrieval; do not infer a TSG101-specific fixation effect (general IHC practice). |
| IF/ICC: what localisation should be compared? | HPA reports mainly cytosolic signal, with additional nucleolar and plasma membrane localisation, in ICC-IF; its cytosol call is Supported (HPA: subcellular ICC-IF). Use that as an IF localisation comparison, allowing for the different readout from chromogenic tissue IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells show no stain (HPA: High in appendix and colon). | A failed detection step or an unsuitable run condition is possible; absence alone cannot identify the failed step (general IHC practice). | Confirm the positive control, detection reagents and the catalog antibody’s IHC-P instructions; review retrieval and antibody dilution within the validated workflow (general IHC practice). |
| The section is uniformly brown, including spaces outside cells. | Nonspecific deposit or detection background can obscure the cellular pattern (general IHC practice). | Compare the primary-antibody omission control; inspect washes, blocking and detection development, then rescore only distinct cellular staining (general IHC practice). |
| Adipocytes stain more strongly than nearby expected positive cells. | That pattern conflicts with HPA’s Not detected adipocyte call and could reflect nonspecific or endogenous signal (HPA: adipocytes Not detected; general IHC practice). | Check omission and known-positive controls under the same detection conditions; avoid assigning the adipocyte signal to TSG101 until the discrepancy is resolved (general IHC practice). |
| Signal looks exclusively nuclear in most cells. | The reported profile is mainly cytoplasmic, although nuclear localisation can vary with cell cycle (UniProt Q99816; HPA: tissue IHC). | Inspect cytoplasmic signal in an HPA-reported positive cell type and compare controls; do not reject a restricted nuclear signal solely because it is nuclear (HPA: High cell types; UniProt Q99816; general IHC practice). |
| Membrane staining is present, but the slide lacks a clear cytoplasmic component. | HPA reports both cytoplasmic and membranous expression; UniProt describes membrane association alongside a mainly cytoplasmic distribution (HPA: tissue IHC; UniProt Q99816). | Check whether staining is cellular and matches expected cell types, then compare a positive control before scoring an isolated membrane-only pattern (general IHC practice). |
| The sample appears negative while the positive control stains. | A true low result is possible, but the supplied HPA profile does not predict every cell in every specimen (HPA: tissue IHC). | Document the cell type, compartment and control result; compare with HPA’s reported High and Not detected cell types, and limit the conclusion to the tested section (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing TSG101 staining
M01233 · IHCImmunohistochemical analysis of paraffin-embedded Rat liver, using the Antibody at 1:500 dilution.
M01233 · IHCImmunohistochemical analysis of paraffin-embedded Human prostate cancer, using the Antibody at 1:1000 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000074319-TSG101/tissue/cerebral+cortex#imid_1411076
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000074319-TSG101/tissue/colon#imid_1411010
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000074319-TSG101/tissue/liver#imid_2185727
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000074319-TSG101/tissue/kidney#imid_1410996
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006161; green = TSG101, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006161; green = TSG101, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TSG101 IHC & IF
🧪Run cerebral cortex first: neuronal cells should stain (HPA: High in neuronal cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the cortex slide, compare stained neurons with neighboring cells that lack signal, without assuming those cells are biologically TSG101-negative.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TSG101 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality, and TSG101 knockout tissue or cells as a biological negative. For chromogenic cortex IHC, block endogenous peroxidase and check for pigment that could be mistaken for stain; assess endogenous biotin if using avidin–biotin detection.
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the selected M01233 paraffin-section caption reports 1:500 in rat liver but leaves the fixative unreported (catalog antibody caption). Retrieval dependency is unreported, so optimize antigen retrieval empirically for paraffin IHC; the evidence does not establish that frozen sections or IF are easier. In cerebral cortex, neuronal lipofuscin can complicate interpretation of chromogenic signal and can autofluoresce in IF (standard histology practice).
HPA tissue IHC evidence for TSG101
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | 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 TSG101 IHC Tips
Troubleshoot TSG101 staining in paraffin sections by checking retrieval, controls, compartment patterns and scoring before interpreting chromogenic signal.
How should I troubleshoot weak TSG101 staining after antigen retrieval?
Use Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min first (page retrieval setting). If staining remains weak, compare retrieved sections with an otherwise identical control section, keeping the catalog antibody at the captioned 1:500 dilution initially (M01233 tissue-IHC caption). Check whether tissue morphology is preserved and whether signal appears in cytoplasm or along membranes, the broad pattern reported for TSG101 (HPA tissue IHC). A milder retrieval trial can help assess tissue damage, but its buffer and duration require validation for this antibody (standard IHC practice). Do not interpret a stronger edge signal alone as improved antigen recovery (standard IHC practice).
Can fixation explain weak or uneven TSG101 staining in paraffin sections?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (M01233 tissue-IHC caption). Record the actual fixative, fixation duration and processing history for each specimen before comparing staining across blocks (standard IHC practice). Compare adjacent sections from blocks processed alike, using the same 1:500 antibody dilution and detection run where appropriate (M01233 tissue-IHC caption; standard IHC practice). If staining varies, inspect preservation and compare retrieval conditions while holding antibody concentration and development time constant (standard IHC practice). Do not assign a TSG101-specific fixation effect from tissue expression patterns or protein features (HPA tissue IHC; UniProt Q99816).
Which TSG101 staining compartments should I accept in tissue?
Expect predominantly cytoplasmic signal, with membranous staining possible in tissue sections (UniProt Q99816 subcellular; HPA tissue IHC). TSG101 associates with early and late endosome membranes and lacks a transmembrane segment, so a purely continuous cell-surface outline deserves scrutiny (UniProt Q99816 subcellular and topology). Nuclear localisation can vary with the cell cycle, while midbody localisation requires interaction with CEP55 during cytokinesis (UniProt Q99816 subcellular). Compare each apparent compartment with cell morphology, a matched negative control and an independently stained section before assigning it to TSG101 (standard IHC practice). Record cytoplasmic, membranous and nuclear signal separately rather than collapsing them into one score (standard IHC practice).
Could an isoform or masked epitope change the staining pattern?
TSG101 has 2 annotated isoforms, but the supplied antibody evidence does not identify which isoform or epitope the catalog antibody recognises (UniProt Q99816 isoforms; M01233 tissue-IHC caption). Its UEV domain spans residues 2–145 and its SB domain spans 322–390; neither location establishes this antibody’s binding site (UniProt Q99816 domains). Annotated modifications include N-acetylalanine at residue 2 and phosphothreonine at 220, without evidence here that either changes staining (UniProt Q99816 modified residues). If retrieval changes signal, document the protocol and compare compartment patterns across matched sections (standard IHC practice). Seek epitope mapping or independent antibody validation before attributing discordance to an isoform or modification (standard IHC practice).
How can IF help resolve ambiguous TSG101 staining?
Use IF/ICC as a separate validation experiment, pairing TSG101 with a marker for the cell type expected in the specimen (standard IF practice). Choose fluorophores in channels with low specimen autofluorescence, and include single-stain controls when assessing overlap (standard IF practice). Permeabilise sufficiently for an epitope facing the cytosol, since TSG101 is mainly cytoplasmic and can associate with endosome membranes without a transmembrane segment (UniProt Q99816 subcellular and topology). The antibody epitope is unspecified, so optimise permeabilisation empirically rather than assuming which side of a membrane it exposes (M01233 tissue-IHC caption; standard IF practice). Compare cytosolic signal with the supported IF localisation while keeping fixation conclusions assay-specific (HPA subcellular; standard IF practice).
How do I reduce diffuse or granular chromogenic background?
First compare the suspect section with a no-primary control and a similarly processed section from the same staining run (standard IHC practice). For peroxidase detection, use a peroxidase block before DAB development and check whether pigment remains in the control (standard chromogenic IHC practice). Adjust blocking, washes and development time one variable at a time, retaining the captioned 1:500 antibody dilution as the initial reference (M01233 tissue-IHC caption; standard IHC practice). True TSG101 can have cytoplasmic and membranous distributions, so diffuse colour alone cannot establish nonspecific binding (HPA tissue IHC). Examine folds, cut edges and damaged regions separately before changing the staining protocol (standard IHC practice).
What is a defensible way to quantify TSG101 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because TSG101 staining may be cytoplasmic or membranous (HPA tissue IHC). For intensity-based analysis, report an H-score on a 0–300 scale, calculated from the percentages of cells at intensities 0–3 (standard IHC scoring practice). Alternatively report the percentage of positive cells or positive-cell density per mm², with the threshold fixed before comparison (standard IHC scoring practice). Normalise counts to eligible cells or measured viable tissue area, and exclude folds and necrotic areas by a prespecified rule (standard IHC scoring practice). Score nuclear signal separately if it is a study endpoint, since its localisation can depend on cell cycle (UniProt Q99816 subcellular).
When is a TSG101-positive IHC result credible?
A credible result has cellular staining consistent with predominantly cytoplasmic, sometimes membranous TSG101 and survives comparison with a no-primary control (UniProt Q99816 subcellular; HPA tissue IHC; standard IHC practice). Check the labelled cell type against tissue morphology; the HPA tissue profile reports high staining in several named cell populations but only medium consistency with RNA expression (HPA tissue IHC). Question staining restricted to section edges, folds or necrotic regions, especially when similar colour appears without primary antibody (standard IHC practice). Residual endogenous peroxidase activity can also produce misleading chromogen, so inspect the peroxidase-block control where relevant (standard chromogenic IHC practice). Treat isolated strong nuclear staining cautiously because TSG101 is mainly cytoplasmic, although nuclear localisation can be cell cycle-dependent (UniProt Q99816 subcellular).
Boster reagents
Best TSG101 / Tumor susceptibility gene 101 protein IHC Antibodies
M01233 has IHC images from paraffin-embedded rat liver and stomach, human prostate cancer, and mouse kidney (M01233 IHC captions); both catalog antibodies list human, mouse, and rat reactivity (catalog reactivity).
Real IHC data
Anti-TSG101 Rabbit Monoclonal Antibody
Cat # M01233
M01233 is the SKU with a rendered IHC card: its captions show paraffin-embedded rat liver and stomach, human prostate cancer, and mouse kidney (M01233 IHC captions). M01233-2 lists IF and human, mouse, and rat reactivity, but has no supplied IHC or IF image for a card (M01233-2 catalog applications/reactivity/image captions).
Which to pick: Choose M01233 for tissue IHC: it lists IHC and has paraffin-section images; the fixative is unreported (M01233 catalog applications and IHC captions). For IF/ICC, M01233 lists both applications, while M01233-2 lists IF only; neither has a supplied IF image (catalog applications and IF image captions). Both are rabbit monoclonals listed as reactive with human, mouse, and rat, but only M01233 has cross-species IHC images (catalog host/title/reactivity; M01233 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q99816 (TS101_HUMAN, Tumor susceptibility gene 101 protein).
- Human Protein Atlas. TSG101 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. TSG101 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoli and plasma membrane..
- Human Protein Atlas. TSG101 antibody validation summary (2 antibodies).
- Expression of TSG101 protein and LSF transcription factor in HPV-positive cervical cancer cells. Oncology letters 2014 — PMC3997686.
- Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury. International journal of molecular medicine 2017 — PMC5593464.
- siRNA against TSG101 reduces proliferation and induces G0/G1 arrest in renal cell carcinoma - involvement of c-myc, cyclin E1, and CDK2. Cellular & molecular biology letters 2019 — PMC6332891.
- Analysis of the Expression of LSF Transcription Factor in the Regulation of Transcription and TSG101 during the Neoplastic Transformation of Endometrial Cells. Cells 2024 — PMC11011417.
- PubMed PMID:9019400 — UniProt-cited evidence.
- PubMed PMID:9867424 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TSG101 (Tumor susceptibility gene 101 protein). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M01233 at 1:50 recommended; image 1:500 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Rat liver tissue; fixative not specified (datasheet M01233); 2. Fixation: Image fixative and duration unreported (datasheet M01233); 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 monoclonal (clone IDG-20) anti-TSG101, 1:50 recommended; image 1:500 (datasheet M01233); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Cell-cycle-dependent nuclear localisation may alter the pattern (UniProt); Isoform / epitope: 2 isoforms; confirm antibody epitope coverage (UniProt); Regulation: Intensity regulation not specified (UniProt). Evidence: UniProt Q99816, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet M01233, PMC3997686, PMC6332891. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TSG101/immunohistochemistry-ihc-guide.