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FHIT / Bis(5'-adenosyl)-triphosphatase · IHC design guide
Design Immunohistochemistry for FHIT
Plan FHIT chromogenic IHC in paraffin sections with the IHC-validated antibody at 0.5–1 μg/mL (datasheet: PB9181). Use HPA cell-level staining to select controls and interpret the generally cytoplasmic tissue pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FHIT 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 | Generally cytoplasmic; cerebellar GLUC-cell nuclei stain (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic staining; high in kidney tubule cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9181) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Vagina |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation and processing consistent across paraffin sections. (selected-SKU IHC image PB9181) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | One 1–147 chain; no annotated isoforms or processing (UniProt) |
Section 1
Recommended FHIT IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet PB9181) accompanies three published FHIT paraffin-section protocols (PMC2409597; PMC2376126; PMC2394260).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet PB9181) |
| Fixation | Image fixative and duration unreported (datasheet PB9181); 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 PB9181); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9181) |
| Primary antibody | Rabbit anti-FHIT, 0.5-1μg/ml (datasheet PB9181) |
| Primary incubation | Overnight at 4 °C (datasheet PB9181) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9181) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FHIT-positive staining in gLUC cells - nucleus of cerebellum (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet PB9181); the published protocols used citrate-based retrieval at pH 6.0 (PMC2409597; PMC2376126; PMC2394260).
Section 2
What Is the Expected FHIT Staining Pattern?
FHIT staining in paraffin tissue sections is generally cytoplasmic, with strong staining reported in kidney tubule cells and several glandular or neuronal cell populations (HPA tissue IHC: Enhanced reliability; medium consistency with RNA). Nuclear staining occurs in cerebellar GLUC cells (HPA tissue IHC). UniProt also lists cytoplasm, mitochondrion and nucleus, and reports no transmembrane segment (UniProt P49789: subcellular location; topology).
| Strong cytoplasmic staining in kidney tubule cells, with limited surrounding background. | This matches a reported high-staining cell population and HPA’s general cytoplasmic IHC profile (HPA tissue IHC: kidney tubules High; general cytoplasmic expression). Judge the signal within identified tubules rather than averaging across the whole section (general IHC practice). |
| A compartment dominates that is unexpected for the tissue, such as membrane-only staining throughout a kidney section. | Treat this as a possible artefact and review morphology and controls before scoring (HPA tissue IHC: general cytoplasmic expression; general IHC practice). Nuclear signal is not automatically wrong: HPA reports high nuclear staining in cerebellar GLUC cells, and UniProt lists a nuclear location (HPA tissue IHC: cerebellum; UniProt P49789: subcellular location). |
| Strong staining appears in vaginal squamous epithelial cells instead of the expected cell population. | Investigate antibody cross-reactivity or endogenous detection activity before calling it FHIT (HPA tissue IHC: vaginal squamous epithelial cells Not detected; general IHC practice). Use the same detection controls and scoring threshold when comparing sections (general IHC practice). |
| Color spreads across stroma, empty spaces or many unrelated cells without clear cell boundaries. | Diffuse background is unsuitable for assigning FHIT to a cell type (general IHC practice). Review blocking, washes and detection controls, then interpret only staining that resolves with tissue morphology (general IHC practice). |
| Kidney tubules show no convincing staining. | A run-wide failure is possible because kidney tubule cells are reported as High (HPA tissue IHC: kidney tubules High). Check tissue preservation, reagent performance and a matched positive control before interpreting another tissue as FHIT-negative (general IHC practice). |
💡Expected FHIT appearanceA convincing positive shows strong, cell-resolved cytoplasmic staining in kidney tubules or other HPA High populations, while widespread compartment-free color or strong staining in vaginal squamous cells warrants investigation (HPA tissue IHC: general cytoplasmic expression; kidney tubules High; vaginal squamous epithelial cells Not detected; general IHC practice).
| Cell type and tissue selection | HPA reports High staining in kidney tubules, thyroid and parathyroid glandular cells, cerebral cortical neurons, rectal endocrine cells and cerebellar GLUC nuclei; vaginal squamous epithelial cells are Not detected (HPA tissue IHC). Select and score the relevant cell population, since adjacent cells need not share its appearance (general IHC practice). |
| Strength of IHC evidence | HPA rates the tissue IHC profile Enhanced but describes only medium consistency between staining and RNA data (HPA tissue IHC: reliability). Two listed antibodies have Enhanced IHC status; a third is Supported (HPA antibodies: HPA018840, HPA018909, CAB002684). Keep those evidence levels distinct when weighing an unexpected result. |
| Compartment and assay context | The tissue IHC profile is generally cytoplasmic, whereas HPA ICC-IF reports approved nucleoli fibrillar center and plasma membrane locations (HPA tissue IHC; HPA subcellular ICC-IF). UniProt lists cytoplasm, mitochondrion and nucleus (UniProt P49789: subcellular location). Resolve apparent disagreement by assay and cell type before transferring an IF pattern to paraffin IHC. |
| Phosphorylation versus total FHIT | UniProt reports phosphotyrosines at residues 114 and 145 and phospho-FHIT in liver and kidney but not brain or lung (UniProt P49789: modified residues; tissue specificity). Those observations do not establish what a total-FHIT IHC antibody detects; interpret phospho-specific staining only with evidence for that antibody’s target (general IHC practice). |
| Topology and processing | UniProt describes a single 1–147 chain, no signal peptide, no propeptide, no transmembrane segment and no listed glycosylation sites (UniProt P49789: processing; topology; glycosylation). A broadly membrane-only paraffin IHC result therefore needs scrutiny, while ICC-IF membrane localization remains separately reported (HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in the positive control | The IHC run may have failed; kidney tubules are reported High, but an individual section still needs interpretable morphology (HPA tissue IHC: kidney tubules High; general IHC practice). | Check control tissue, antigen retrieval, primary antibody dilution and chromogenic detection against the validated IHC workflow; repeat with a working positive control (general IHC practice). |
| All cells or tissue spaces look stained | Diffuse background can reflect incomplete blocking, inadequate washes or detection activity rather than cell-resolved FHIT (general IHC practice). | Review a no-primary control, blocking and wash steps; score only signal that follows identifiable cells (general IHC practice). |
| Vaginal squamous cells stain strongly | This conflicts with their Not detected HPA result; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC: vaginal squamous epithelial cells Not detected; general IHC practice). | Compare a matched positive tissue and no-primary control, then reassess whether the signal follows the expected cell compartment (HPA tissue IHC: general cytoplasmic expression; general IHC practice). |
| Kidney signal is chiefly membranous or nuclear | That distribution differs from HPA’s general cytoplasmic tissue IHC profile, although nuclear FHIT is reported in another cell population (HPA tissue IHC: general cytoplasmic expression; cerebellar GLUC nuclei). | Confirm cell identity and counterstained morphology, review controls, and avoid assigning the pattern from ICC-IF to this paraffin section (HPA subcellular ICC-IF; general IHC practice). |
| Only a few cells stain in a mixed tissue | FHIT staining varies by cell population: HPA reports High, Medium, Low and Not detected observations across the listed tissues (HPA tissue IHC). | Score the named cell type separately and compare it with that population’s HPA observation; do not label the entire section positive or negative from a small subset (HPA tissue IHC; general IHC practice). |
| Does IF/ICC require the same pattern as paraffin IHC? | HPA ICC-IF reports nucleoli fibrillar center and plasma membrane, while tissue IHC reports general cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret IF/ICC using its own assay and cell context; this paraffin IHC interpretation does not establish an IF/ICC protocol (HPA subcellular ICC-IF; general IHC practice). |
Experiment evidence
IHC & IF images showing FHIT staining
PB9181 · IHCIHC analysis of FHIT using anti-FHIT antibody (PB9181). FHIT was detected in a paraffin-embedded section of human mammary cancer tissue. Hea
PB9181 · IFIF analysis of FHIT using anti-FHIT antibody (PB9181). FHIT was detected in immunocytochemical section of U20S cells. Enzyme antigen retriev
A01200 · IFImmunofluorescence of FHIT in HeLa cells with FHIT antibody at 5 μg/mL.
M01200 · IFIF analysis of FHIT using anti-FHIT antibody (M01200). FHIT was detected in immunocytochemical section of MCF7 cells. Enzyme antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000189283-FHIT/tissue/cerebral+cortex#imid_816464
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000189283-FHIT/tissue/colon#imid_816382
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000189283-FHIT/tissue/liver#imid_816399
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000189283-FHIT/tissue/kidney#imid_816384
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA018909; green = FHIT, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA018909; green = FHIT, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FHIT IHC & IF
🧪Run kidney first: tubular cells should stain strongly (HPA: High in cells in tubules). Use vagina as the negative tissue; its squamous epithelial cells should remain unstained (HPA: Not detected). On the kidney slide, assess cells outside the tubules as an internal background reference, without assuming a specific cell population is FHIT-negative (HPA: kidney row identifies tubular cells only).
Positive control tissue: Cerebellum (GLUC cells - nucleus, HPA High)
Negative control tissue: Vagina (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FHIT in HEK293, U-251MG, U2OS, with annotated localisation: Nucleoli fibrillar center (approved), Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a rabbit IgG isotype control matched to the primary antibody, and FHIT-knockout material as a biological negative (PB9181 caption: rabbit primary; standard IHC practice). For kidney, quench endogenous peroxidase and check endogenous biotin background if using the caption’s avidin–biotin detection system (PB9181 caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9181 paraffin-section caption does not state a fixative (PB9181 caption). The reported IHC procedure uses heat retrieval in EDTA at pH 8.0; its result does not establish whether retrieval is essential (PB9181 caption). The supplied evidence does not establish whether frozen sections or IF are easier for FHIT; endogenous biotin may complicate avidin–biotin detection in kidney (standard IHC practice).
HPA tissue IHC evidence for FHIT
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Cerebellum | GLUC cells - nucleus | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FHIT IHC Tips
Troubleshoot FHIT staining in paraffin sections by checking retrieval, cellular distribution, controls, and scoring before interpreting loss of expression.
What retrieval should I try first when FHIT staining is weak?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet PB9181). The selected tissue image used this retrieval before overnight incubation with 1 μg/ml catalog antibody at 4°C, so reproduce those conditions when troubleshooting that workflow (caption PB9181). If signal remains weak, compare a modestly longer heating interval or citrate at pH 6.0 on adjacent sections, while keeping detection and exposure to chromogen constant (standard IHC practice). Include a known staining tissue, such as kidney tubules, and a no-primary control so a stronger signal can be weighed against increased background (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or patchy FHIT staining?
Target-specific FHIT sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not report its fixative (caption PB9181). Record fixation method, duration, and processing age for each specimen before attributing weak staining to the antigen or antibody (standard IHC practice). Compare sections processed under the same conditions, using the caption's EDTA pH 8.0 retrieval and 1 μg/ml primary incubation as a starting point (caption PB9181). Examine tissue morphology and staining controls together; uneven preservation can make a regional loss of chromogen difficult to interpret as biological loss (standard IHC practice).
Which cellular compartments should count as plausible FHIT staining?
Expect a broadly cytoplasmic tissue pattern, while allowing nuclear and mitochondrial localisation described for FHIT (HPA tissue IHC; UniProt P49789). A separate cell-imaging assessment reports signal at the nucleoli fibrillar center and plasma membrane, so compartment claims should specify which assay supports them (HPA subcellular). FHIT has no transmembrane segment, and the record supplies no membrane-facing epitope assignment (UniProt P49789 topology). Score cytoplasm and nucleus separately where morphology permits, and compare candidate signal with no-primary and tissue controls before treating a sharp surface outline as specific FHIT staining (standard IHC practice).
How can epitope uncertainty affect FHIT IHC interpretation?
The supplied record lists 0 annotated FHIT isoforms and a 147-residue chain, but it does not identify the catalog antibody's epitope (UniProt P49789; caption PB9181). Its HIT domain spans residues 2–109, with reported phosphotyrosines at 114 and 145; these features cannot establish what this antibody recognizes (UniProt P49789). Keep retrieval and primary-antibody conditions fixed when comparing specimens, because accessibility changes can mimic expression differences (standard IHC practice). If an epitope-dependent explanation matters, obtain the antibody's mapped immunogen or epitope and test specificity with an appropriate independent control before assigning a phospho-specific or variant-specific pattern (standard IHC practice).
How should I assess FHIT by multiplex immunofluorescence?
For a separate IF experiment, pair FHIT with a validated marker identifying the cell population being assessed, such as kidney tubular cells when studying the HPA-positive kidney pattern (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue in each channel before assigning signal, because tissue autofluorescence can imitate weak staining (standard IF practice). FHIT is reported in intracellular compartments and has no transmembrane segment, so permeabilise when the antibody epitope is intracellular; its precise epitope side is unreported (UniProt P49789 topology; standard IF practice). Optimise permeabilisation and exposure with single-stain and no-primary controls, and report IF localisation independently of chromogenic IHC (standard IF practice).
What should I check when DAB obscures FHIT staining?
First compare the stained section with a no-primary control to separate antibody-associated signal from detection-system or tissue background (standard IHC practice). The selected workflow uses a biotinylated secondary, streptavidin-biotin complex, and DAB; check the corresponding control for endogenous biotin or peroxidase contribution (caption PB9181; standard IHC practice). Apply a peroxidase block and appropriate protein block, then titrate the 1 μg/ml primary starting condition or shorten DAB development if diffuse colour persists (caption PB9181; standard IHC practice). Review section edges and damaged areas separately from intact cells, and preserve a readable counterstain so cellular localisation can be judged (standard IHC practice).
How should I quantify heterogeneous FHIT staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA describes general cytoplasmic tissue expression while UniProt also lists nuclear localisation (HPA tissue IHC; UniProt P49789). Record the percentage of positive eligible cells and intensity categories, then calculate an H-score as the sum of each category's percentage multiplied by its intensity grade (standard IHC practice). If measuring positive-cell density, report cells per mm² of viable tissue and the area sampled (standard IHC practice). Normalise comparisons to the same cell type, viable area, staining run, and control range; exclude necrosis, folds, and cut edges by prespecified rules (standard IHC practice).
When does apparent FHIT loss or positivity warrant caution?
Treat a candidate positive as more credible when chromogen follows intact cells in a plausible compartment and the same run's no-primary control is clear (UniProt P49789; standard IHC practice). HPA reports strong staining in kidney tubules and neuronal cells, but no detected staining in vaginal squamous epithelial cells; compare like cell populations before calling loss (HPA tissue IHC). Be cautious with edge-only colour, necrotic regions, or signal persisting without primary antibody, which can reflect processing or endogenous enzyme activity (standard IHC practice). FHIT is reported in cytoplasm, mitochondria, and nucleus, while a separate subcellular assay also reports plasma membrane localisation; an unexpected pattern needs an independent specificity check (UniProt P49789; HPA subcellular; standard IHC practice).
Boster reagents
Best FHIT / Bis(5'-adenosyl)-triphosphatase IHC Antibodies
Anti-FHIT antibodies have human paraffin-section IHC and human-cell IF images (catalog figure captions). Catalog reactivity also lists rat for PB9181 and mouse for A01200 (catalog reactivity).
Real IHC data
Anti-FHIT Antibody ®
Cat # PB9181
Real IF data
Anti-FHIT Antibody
Cat # A01200
Real IF data
Anti-FHIT Antibody ® (monoclonal, 26H7)
Cat # M01200
PB9181 shows IHC in a paraffin section of human mammary cancer tissue and IF in U20S cells (PB9181 image captions). A01200 shows IF in HeLa cells, while M01200 shows IF in MCF7 cells (A01200 and M01200 image captions).
Which to pick: Choose PB9181 for human paraffin-section chromogenic IHC: its figure documents EDTA retrieval at pH 8.0, 1 μg/ml primary antibody and DAB detection; the fixative is unreported (PB9181 IHC image caption). For human-cell IF/ICC, M01200 is a mouse monoclonal listed for both applications, while A01200 is a rabbit antibody listed for IF (catalog applications and antibody details; M01200 and A01200 IF image captions). For cross-species planning, PB9181 lists rat reactivity and A01200 lists mouse reactivity, while the supplied PB9181 IHC figure documents human tissue only (catalog reactivity; PB9181 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49789 (FHIT_HUMAN, Bis(5'-adenosyl)-triphosphatase).
- Human Protein Atlas. FHIT tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FHIT subcellular location (ICC-IF): Localized to the nucleoli fibrillar center and plasma membrane..
- Human Protein Atlas. FHIT antibody validation summary (3 antibodies).
- Fhit expression in human gastric adenomas and intramucosal carcinomas: correlation with Mlh1 expression and gastric phenotype. British journal of cancer 2004 — PMC2409597.
- Reduced Fhit expression is associated with mismatch repair deficiency in human advanced colorectal carcinoma. British journal of cancer 2002 — PMC2376126.
- Roles of Fhit and p53 in Taiwanese surgically treated non-small-cell lung cancers. British journal of cancer 2003 — PMC2394260.
- Nit1 and Fhit tumor suppressor activities are additive. Journal of cellular biochemistry 2009 — PMC2762784.
- PubMed PMID:8598045 — UniProt-cited evidence.
- PubMed PMID:9012482 — UniProt-cited evidence.
- PubMed PMID:24722188 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FHIT (Bis(5'-adenosyl)-triphosphatase). Expected localisation Generally cytoplasmic; cerebellar GLUC-cell nuclei stain (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9181). Validated IHC antibody: PB9181 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (GLUC cells - nucleus); negative Vagina. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet PB9181); 2. Fixation: Image fixative and duration unreported (datasheet PB9181); 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 PB9181); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9181); 8. Primary antibody: Rabbit anti-FHIT, 0.5-1μg/ml (datasheet PB9181); 9. Primary incubation: Overnight at 4 °C (datasheet PB9181). Watch for: Fixation: Keep fixation and processing consistent across paraffin sections. (selected-SKU IHC image PB9181); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: One 1–147 chain; no annotated isoforms or processing (UniProt); Regulation: Expression varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt P49789, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoli fibrillar center and plasma membrane., Datasheet PB9181, PMC2409597, PMC2376126. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FHIT/immunohistochemistry-ihc-guide.