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FHIT / Bis(5'-adenosyl)-triphosphatase · IHC design guide
Design Immunohistochemistry for FHIT
Plan chromogenic FHIT IHC on paraffin sections using the catalog antibody at 0.5–1 µg/mL (datasheet PB9181). Select comparison tissues and score cytoplasmic staining by cell type using the reported 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 | General cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across multiple cell types (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 consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Vaginal squamous cells may show no signal (HPA tissue IHC) | |
| Regulation | FHIT levels vary by cell type (HPA tissue IHC) | |
| Isoform / epitope | One chain, residues 1–147; no isoforms reported (UniProt) |
Section 1
Recommended FHIT IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9181) is accompanied by four published FHIT protocols (PMC2409597; PMC2376126; PMC2394260; PMC2762784).
| 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 pH 8.0 for the catalog antibody (datasheet: PB9181); use each paper’s stated retrieval conditions when reproducing its protocol.
Section 2
What Is the Expected FHIT Staining Pattern?
In paraffin sections, expect chiefly cytoplasmic FHIT staining (HPA: general cytoplasmic IHC expression). Kidney tubular cells, cerebral cortical neurons, and thyroid and parathyroid glandular cells are useful high staining examples (HPA: High in each). Nuclear staining can occur in cerebellar GLUC cells (HPA: High nuclear staining). FHIT has no transmembrane segment, although UniProt also lists nuclear and mitochondrial locations (UniProt P49789: topology and subcellular location). HPA rates its tissue IHC evidence Enhanced, with medium agreement between staining and RNA data (HPA: reliability).
| Distinct cytoplasmic staining in kidney tubules or thyroid glandular cells, with recognizable cell boundaries. | This fits the reported tissue pattern and offers a practical positive reference (HPA: High in kidney tubular and thyroid glandular cells). Judge the named cells separately from surrounding tissue; a whole-section impression can hide a cell-specific result (general IHC practice). |
| Nuclear staining in cerebellar GLUC cells, or nuclear signal alongside cytoplasmic signal elsewhere. | Cerebellar GLUC nuclear staining is reported as High (HPA: cerebellum). UniProt also lists the nucleus (UniProt P49789: subcellular location). Nuclear signal alone in an unlisted cell type needs control-based review; the cerebellar observation does not establish that every tissue should stain in nuclei. |
| Signal confined to extracellular deposits, luminal material, or a crisp membrane rim in tissue IHC. | Treat this as a possible artefact and compare it with positive-cell morphology and controls (general IHC practice). FHIT lacks a transmembrane segment (UniProt P49789: topology), while HPA reports a plasma membrane location in ICC-IF, so a membrane rim alone cannot prove either specificity or error (HPA: subcellular ICC-IF). |
| Strong color in cells outside the expected positive population, including across a control section. | Consider cross-reactivity or endogenous detection activity before calling FHIT positive (general IHC practice). HPA's levels refer to specified cell types, such as High in rectal endocrine cells and not detected in vaginal squamous epithelial cells (HPA: tissue IHC); they do not assign one intensity to every cell in those tissues. |
| Diffuse haze throughout the section, or no signal in kidney tubular cells used as a positive reference. | Diffuse haze obscures compartment and cell-type assessment and suggests background (general IHC practice). An absent kidney tubular signal conflicts with a reported High population (HPA: kidney); check the run and antibody conditions before interpreting other negative fields. |
💡Expected FHIT appearanceCall a convincing positive when distinct, chiefly cytoplasmic color marks the reported cell population, such as High staining in kidney tubules (HPA: kidney; HPA: general cytoplasmic profile); nuclear staining in cerebellar GLUC cells is also reported (HPA: cerebellum), whereas uniform haze or color detached from cell morphology is suspect (general IHC practice).
| Tissue and cell choice | Kidney tubular cells and cerebral cortical neurons are reported High, while vaginal squamous epithelial cells are not detected (HPA: tissue IHC). Use the named populations when comparing sections; a negative cell population is a contextual reference, not proof that every unstained field is technically valid (general IHC practice). |
| Antibody validation | HPA018840 and HPA018909 have Enhanced IHC status; CAB002684 has Supported IHC status (HPA: antibodies). HPA describes overall tissue IHC reliability as Enhanced with medium staining/RNA consistency (HPA: reliability). Record which antibody produced the image when comparing patterns across runs (general IHC practice). |
| Subcellular context | HPA summarizes tissue IHC as generally cytoplasmic but reports approved nucleoli fibrillar center and plasma membrane locations in ICC-IF (HPA: tissue IHC; HPA: subcellular ICC-IF). These are observations from different assays; do not require an ICC-IF membrane pattern in paraffin IHC. |
| Phosphorylation and antibody specificity | UniProt lists phosphotyrosines at residues 114 and 145 and reports tissue-dependent phospho-FHIT observations (UniProt P49789: modified residues and tissue specificity). These facts do not show whether an unspecified IHC antibody distinguishes phosphorylated FHIT; interpret its staining as total FHIT only if its own specificity documentation supports that call. |
| IF/ICC Q&A: Should the paraffin IHC pattern be copied into IF/ICC? | No. HPA reports nucleoli fibrillar center and plasma membrane as approved ICC-IF locations, with images from HEK293, U-251MG, and U2OS (HPA: subcellular ICC-IF). Use the separate IF/ICC guide for that application; the tissue IHC profile alone cannot predict the compartment pattern in those cells. |
| Situation | Likely cause | Next action |
|---|---|---|
| No color in kidney tubular cells and no convincing signal elsewhere. | The run may have failed or the chosen antibody conditions may be unsuitable (general IHC practice); kidney tubular cells are reported High (HPA: kidney). | Check a known-positive section, reagent order, retrieval step, and detection controls within the antibody's IHC-P instructions (general IHC practice). Do not score a test section as FHIT-negative until the positive reference stains. |
| Diffuse color covers both cells and open tissue spaces. | Nonspecific background or endogenous detection activity can obscure a cell-localized result (general IHC practice). | Compare a no-primary control, review blocking and washes, and inspect whether signal follows recognizable cells (general IHC practice). Reassess the slide after the background source is identified; haze does not match HPA's general cytoplasmic profile (HPA: tissue IHC). |
| Only a nuclear signal appears in an unlisted tissue or cell population. | FHIT can localize to the nucleus (UniProt P49789: subcellular location), but HPA's explicit High nuclear tissue example is cerebellar GLUC cells (HPA: cerebellum). | Check morphology and controls, then compare the same antibody in a reported positive population (general IHC practice; HPA: tissue IHC). Avoid declaring the result false solely because it is nuclear. |
| Bright staining appears in vaginal squamous epithelial cells. | HPA reports FHIT as not detected in that named cell population (HPA: vagina); cross-reactivity or detection background is possible (general IHC practice). | Review a no-primary control and compare the antibody in a reported positive tissue (general IHC practice; HPA: tissue IHC). Treat disagreement as a validation question, not proof of a new FHIT distribution. |
| A membrane rim dominates the paraffin section. | HPA's tissue summary is generally cytoplasmic (HPA: tissue IHC), although plasma membrane localization is approved in ICC-IF (HPA: subcellular ICC-IF). The two observations alone cannot resolve this slide. | Inspect whether cytoplasmic signal accompanies the rim and whether controls show the same outline (general IHC practice). Report the compartment observed rather than assigning an ICC-IF localization to tissue IHC. |
| Signal differs sharply between two antibodies on adjacent sections. | Antibodies have distinct HPA IHC validation statuses: HPA018840 and HPA018909 are Enhanced, while CAB002684 is Supported (HPA: antibodies). That status does not identify the cause of a specific discordant slide. | Document antibody identity, compare the named positive cell populations and controls, and review each antibody's IHC-P instructions (general IHC practice; HPA: tissue IHC). Keep the discrepant result unresolved until the comparison supports an interpretation. |
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 kidney cells in tubules). Use vagina as the negative tissue (HPA: Not detected in squamous epithelial cells); on the kidney slide, cells serving as internal negatives should show only background staining, but their FHIT-negative status must be verified rather than assumed.
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 no-primary (secondary-only), host-matched rabbit IgG isotype, and an FHIT knockout specimen as specificity controls (PB9181 caption: rabbit primary; standard IHC practice). Check kidney sections for endogenous peroxidase and biotin background before interpreting DAB signal (PB9181 caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: The paraffin-section caption reports heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; the fixative and any FHIT-specific fixation window or effect are unreported (PB9181 caption). The supplied evidence does not establish whether frozen sections or IF are easier for FHIT (PB9181 caption: paraffin IHC; HPA: ICC-IF images). Endogenous biotin in kidney tubules can complicate avidin–biotin detection (PB9181 caption: SABC; 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, compartment, cell type, controls and scoring before interpreting weak or absent signal.
What should I change when FHIT staining is weak after antigen retrieval?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9181). The selected tissue image used this retrieval before overnight incubation at 4°C with 1 μg/ml primary antibody (caption PB9181). Check that sections remain covered by buffer throughout heating and cooling, then compare staining with a kidney tubule control processed in the same run (HPA: high in kidney tubule cells). If signal remains weak, compare a second retrieval condition on adjacent sections as a fallback, changing only that variable. Judge improvement by clearer staining in the expected cells without increased staining in the negative control (general IHC practice).
Could fixation explain weak FHIT staining in my paraffin sections?
FHIT-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state its fixative (caption PB9181). Record the fixative, time in fixative and tissue thickness for each specimen, then compare sections processed together using the same retrieval and detection settings (general IHC practice). Include a reference section with known FHIT staining, such as kidney tubules, to distinguish a specimen-wide processing problem from a genuinely weak sample (HPA: high in kidney tubule cells). If an overfixed sample is suspected, test retrieval conditions on adjacent sections while retaining the documented EDTA pH 8.0 condition as the reference (datasheet PB9181). Do not assign a fixation effect to FHIT from staining patterns alone (general IHC practice).
Which compartments should count as plausible FHIT staining in IHC?
Expect a predominantly cytoplasmic tissue pattern, while allowing nuclear or mitochondrial staining as biologically plausible (HPA: general cytoplasmic expression; UniProt P49789: cytoplasm, mitochondrion and nucleus). FHIT has no transmembrane segment, so a sharply exclusive membrane rim needs additional validation before it is scored as FHIT (UniProt P49789: topology). An independent subcellular dataset reports nucleoli fibrillar center and plasma membrane localisation, which makes compartment calls dependent on assay context (HPA: subcellular localisation). Compare compartments within clearly identified cells and against the same run’s controls, rather than treating every brown deposit as intracellular signal (general IHC practice). Record cytoplasmic, nuclear and membrane patterns separately when they differ across the specimen (general IHC practice).
How can epitope choice affect my FHIT IHC result?
The supplied FHIT record lists one chain spanning residues 1–147 and zero isoforms, so it does not support an isoform-specific interpretation (UniProt P49789: processing and isoforms). Its HIT domain spans residues 2–109, with reported phosphotyrosines at 114 and 145 (UniProt P49789: domains and modified residues). Check the catalog antibody’s documented immunogen or epitope before attributing a staining difference to phosphorylation; no epitope position is supplied here (general IHC practice). Compare adjacent sections using the same retrieval and a validated control if changing antibodies, and score only reproducible cellular patterns (general IHC practice). Do not infer a particular modification from total-FHIT chromogenic staining without a modification-specific reagent (general IHC practice).
How should I investigate FHIT localisation with multiplex immunofluorescence?
Treat IF as a separate validation exercise: the supplied tissue protocol establishes paraffin-section chromogenic IHC, while the subcellular record lists IF images in HEK293, U-251MG and U2OS (caption PB9181; HPA: subcellular images). Pair FHIT with a marker of the cell type being assessed, such as a validated neuronal marker when examining cerebral cortex, where neuronal staining is reported high (HPA: cerebral cortex neuronal cells). Choose spectrally separated fluorophores and measure an unstained section so tissue autofluorescence is not mistaken for weak FHIT signal (general IF practice). FHIT lacks a transmembrane segment; permeabilise for intracellular epitopes and verify the antibody’s actual epitope before interpreting any membrane-associated signal (UniProt P49789: topology; general IF practice).
How do I reduce diffuse brown background without losing FHIT signal?
First compare the stained section with a no-primary control and inspect whether brown deposit tracks cells or tissue edges (general IHC practice). The selected protocol used 10% goat serum block, 1 μg/ml primary antibody, a biotinylated secondary and DAB detection (caption PB9181). If background is broad, titrate primary concentration and shorten detection development while holding the EDTA pH 8.0 retrieval constant (datasheet PB9181; general IHC practice). Include a peroxidase block and assess endogenous biotin when using biotin-based detection; these are general chromogenic controls, not FHIT-specific findings (general IHC practice). Retain a positive reference section to ensure cleaner slides still show the expected cellular pattern (HPA: high in kidney tubule cells).
How should I score heterogeneous FHIT staining across a tissue section? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and eligible cell population before scoring because FHIT tissue staining is generally cytoplasmic, with nuclear staining reported in cerebellar GLUC cells (HPA: tissue IHC). For each annotated region, record the percentage of positive eligible cells and intensity grades 0–3; their weighted sum gives an H-score from 0–300 (general IHC practice). Alternatively, report positive-cell density per mm² when discrete cells can be counted reliably (general IHC practice). Normalise counts to evaluable tissue area or the total eligible cell count, and exclude folds, necrosis and section edges using the same rules for every sample (general IHC practice). Keep cytoplasmic and nuclear scores separate if both are measured (general IHC practice).
When is apparent FHIT positivity likely to be an artefact?
A convincing result follows cell identity and a reproducible compartment pattern: FHIT is generally cytoplasmic in tissue, and kidney tubule cells show high staining (HPA: tissue IHC). Treat isolated edge staining, deposits over necrosis or signal in a no-primary control as possible processing or detection artefacts (general IHC practice). Check unexpected cell types against the reference pattern; squamous epithelial cells in vagina were reported as not detected in the supplied tissue dataset (HPA: vagina). A membrane-only pattern requires care because FHIT has no transmembrane segment, although an independent subcellular dataset reports plasma membrane localisation (UniProt P49789: topology; HPA: subcellular localisation). Verify questionable DAB signal with peroxidase controls and repeat staining before assigning biological meaning (general IHC practice).
Boster reagents
Best FHIT / Bis(5'-adenosyl)-triphosphatase IHC Antibodies
Anti-FHIT antibodies have IHC data from human mammary cancer paraffin sections (PB9181 image caption) and IF data from human cell samples (A01200, M01200 image captions). Rat and mouse reactivity are catalog-listed (catalog: PB9181, A01200).
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 has IHC data from a human mammary cancer paraffin section and is listed for IHC and IF (PB9181 image caption; catalog: PB9181 applications). A01200 has IF data from HeLa cells, while M01200 has IF data from MCF7 cells and is listed for IF/ICC (A01200 and M01200 image captions; catalog: M01200 applications).
Which to pick: Choose PB9181 for paraffin-section IHC: its IHC caption documents heat retrieval in EDTA at pH 8.0 and primary antibody at 1 μg/ml; the fixative is unreported (PB9181 image caption). For IF/ICC, M01200 is a mouse monoclonal listed for both applications, while A01200 is a rabbit antibody listed for IF (catalog: M01200 clone, host and applications; catalog: A01200 host and applications). For cross-species planning, PB9181 lists human and rat reactivity, and A01200 lists human and mouse reactivity; the cited IHC and IF figures show human samples (catalog: PB9181 and A01200 reactivity; PB9181 and A01200 image captions).
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 General cytoplasmic tissue staining (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 consistent across sections (standard IHC practice; not target-specific); Caveat: Vaginal squamous cells may show no signal (HPA tissue IHC); Isoform / epitope: One chain, residues 1–147 ; no isoforms reported (UniProt); Regulation: FHIT levels vary by 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.