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IFT74 / Intraflagellar transport protein 74 homolog · IHC design guide
Design Immunohistochemistry for IFT74
Plan chromogenic IHC for IFT74 in paraffin sections using ciliated epithelia and late spermatids as reference patterns (HPA tissue IHC). The guide covers fixation consistency and scoring membranous and cytoplasmic staining (HPA tissue IHC), with a catalog antibody tested at 2–5 μg/ml (datasheet A08931-2).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IFT74 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 | Cilia and basal bodies (UniProt); membranous and cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Membranous in ciliated epithelia; cytoplasmic in other tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08931-2) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Cytoplasmic staining also occurs in other tissues (HPA tissue IHC) | |
| Regulation | Staining varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | Two isoforms; no transmembrane segment; epitope coverage unknown (UniProt) |
Section 1
Recommended IFT74 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A08931-2). The published zebrafish retinal cryosection method reports staining conditions but no IFT74 primary-antibody dilution (PMC8431285).
| Sample | Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A08931-2) |
| Fixation | Image fixative and duration unreported (datasheet A08931-2); 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 A08931-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08931-2) |
| Primary antibody | Rabbit anti-IFT74, 2-5 μg/ml (datasheet A08931-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08931-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08931-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IFT74-positive staining in elongated or late spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Membranous expression in ciliated cells in fallopian tube and respiratory epithelia. Cytoplasmic expression in several other tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A08931-2). The published cryosection method used citrate retrieval for connecting-cilia staining (PMC8431285).
Section 2
What Is the Expected IFT74 Staining Pattern?
IFT74 is a ciliary and cytoplasmic vesicle protein with no transmembrane segment (UniProt Q96LB3). In paraffin sections, expect membranous staining in ciliated respiratory and fallopian tube cells, cytoplasmic staining in several other tissues, and strong staining in late spermatids (HPA tissue IHC). HPA rates its tissue IHC profile Approved, pending external verification (HPA tissue IHC).
| Strong staining in elongated or late spermatids; medium staining in bronchial respiratory epithelium or kidney tubules (HPA tissue IHC). | This distribution fits the reported positive cells and levels (HPA tissue IHC). In ciliated epithelia, look for membranous staining; other positive tissues may show cytoplasmic staining (HPA tissue IHC). Judge each compartment against its own tissue context rather than expecting one pattern everywhere. |
| Predominantly nuclear staining, with no corresponding ciliated-cell or cytoplasmic pattern. | A dominant nuclear pattern does not match the reported ciliary, basal body, or vesicular locations (UniProt Q96LB3) or the tissue IHC profile (HPA tissue IHC). Treat it as suspect, review morphology and detection controls, and seek independent confirmation before scoring it as IFT74. |
| Strong staining in adipocytes, marrow hematopoietic cells, or esophageal squamous epithelium. | These cell types are reported as not detected (HPA tissue IHC). Check whether staining follows the cells or instead pools over pigment, debris, or tissue edges. Persistent cell-associated signal could reflect cross-reactivity or endogenous detection activity; confirm with appropriate controls. |
| Diffuse color across the section, including stroma and areas without recognizable positive cells. | The reported IHC pattern is cell and compartment dependent (HPA tissue IHC). Uniform haze prevents reliable scoring of those features. Inspect the negative reagent control and the distribution of background before interpreting weak cytoplasmic color as target staining. |
| No staining in elongated or late spermatids, or no discernible signal in a positive bronchial section. | Late spermatids are reported High and bronchial respiratory epithelial cells Medium (HPA tissue IHC). A blank positive specimen leaves assay performance unresolved. Confirm that the relevant cells are present and intact, then review detection, antibody dilution, and the controls. |
💡Expected IFT74 appearanceCall a result positive when signal matches its cell type and compartment: strong in late spermatids, or membranous in ciliated respiratory epithelium, with other reported tissues often cytoplasmic (HPA tissue IHC); dominant nuclear color or uniform tissue-wide haze is suspect (UniProt Q96LB3; HPA tissue IHC).
| Cell and tissue context (HPA tissue IHC) | Reported levels range from High in late spermatids to Medium in bronchial epithelium and kidney tubules, while several sampled cell types are not detected (HPA tissue IHC). Score the expected cells rather than averaging color across an entire section. |
| Subcellular context (UniProt Q96LB3; HPA tissue IHC) | IFT74 localizes along primary cilia and near the basal body or centriole (UniProt Q96LB3). Tissue IHC also reports membranous staining in ciliated cells and cytoplasmic staining elsewhere (HPA tissue IHC). These patterns need not look identical. |
| Antibody evidence (HPA antibody validation; HPA tissue IHC) | Two listed rabbit polyclonal antibodies have Approved IHC status, while the overall tissue profile remains pending external verification (HPA antibody validation; HPA tissue IHC). Approval supports using the reported pattern as a reference, not treating every colored structure as confirmed IFT74. |
| IF/ICC interpretation (HPA subcellular; HPA antibody validation) | For the separate IF/ICC guide, HPA reports supported Golgi, centriolar satellite, and basal body localization plus approved cytosolic localization (HPA subcellular). The listed ICC assessment for HPA026684 is Uncertain (HPA antibody validation), so an IF image needs its own validation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive testis section is blank. | The reported High signal in elongated or late spermatids is absent (HPA tissue IHC); the reason cannot be assigned from the slide alone. | Check that late spermatids are present, then review the reagent control, chromogenic detection, antibody dilution, and the assay's documented IHC procedure. Repeat with an appropriate positive section if the run control failed. |
| Kidney tubules look weak or variable. | Tubular cells are reported at a Medium level (HPA tissue IHC); weak signal alone does not establish assay failure. | Score intact tubular cells against background and a stronger reported positive, such as late spermatids (HPA tissue IHC). Confirm the tissue and cell identity before changing staining conditions. |
| Diffuse chromogen obscures ciliated-cell borders. | Section-wide background can hide the reported membranous pattern in ciliated epithelia (HPA tissue IHC). | Review negative reagent controls, blocking, wash stringency, and detection exposure as general IHC checks. Reassess the ciliated cells after background is reduced; do not score diffuse haze as localization. |
| Color is concentrated in nuclei or tissue edges. | That distribution is inconsistent with reported IFT74 locations (UniProt Q96LB3; HPA tissue IHC) and may represent nonspecific deposition. | Inspect section morphology and negative controls, then compare another reported positive compartment (HPA tissue IHC). Require reproducible cell-associated staining before assigning the signal to IFT74. |
| A reported negative cell type stains strongly. | Adipocytes, marrow hematopoietic cells, and esophageal squamous cells are listed as not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible. | Confirm the cell type and compare negative reagent and detection controls. If signal persists, seek confirmation with an independently validated antibody before changing the expected pattern. |
| What should an IF/ICC image show? | IF/ICC has separate localization evidence: supported Golgi, centriolar satellite, and basal body signals, with approved cytosolic signal (HPA subcellular). | Use the separate IF/ICC guide for imaging decisions. Interpret an image alongside the antibody's ICC status; HPA026684 is marked Uncertain for ICC (HPA antibody validation). |
Experiment evidence
IHC & IF images showing IFT74 staining
A08931-2 · IHCIHC analysis of IFT74 using anti-IFT74 antibody (A08931-2). IFT74 was detected in a paraffin-embedded section of human esophageal squamous c
A08931-2 · IHCIHC analysis of IFT74 using anti-IFT74 antibody (A08931-2). IFT74 was detected in a paraffin-embedded section of human lung cancer tissue. H
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000096872-IFT74/tissue/cerebral+cortex#imid_6540733
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000096872-IFT74/tissue/colon#imid_6499950
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000096872-IFT74/tissue/liver#imid_6499951
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000096872-IFT74/tissue/kidney#imid_6499952
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026684; green = IFT74, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026684; green = IFT74, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IFT74 IHC & IF
🧪Run testis first and score elongated or late spermatids, the HPA High cell population (HPA: High in elongated or late spermatids). Use adipose tissue as a negative tissue and assess its adipocytes (HPA: Not detected in adipocytes); on the testis slide, treat cells outside the scored spermatid population as internal background comparators, without assuming they are IFT74-negative (HPA: testis cell-type result).
Positive control tissue: Testis (Elongated or late spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IFT74 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Golgi apparatus (supported), Centriolar satellite (supported), Basal body (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-matched nonimmune rabbit IgG or isotype controls, matching the primary antibody’s clonality where applicable (selected-SKU caption: rabbit primary and anti-rabbit secondary). Use verified IFT74-knockout material as a biological negative, or an immunizing-peptide block if the peptide is available; quench endogenous peroxidase and compare testis background with the no-primary slide before interpreting HRP/DAB staining (selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). That caption uses heat retrieval in EDTA at pH 8.0, so assess retrieval when transferring the method to testis; neither a requirement for that condition nor an advantage for frozen sections or IF is established by the supplied evidence (selected-SKU caption: EDTA retrieval; HPA: ICC-IF images). In testis, check that signal assigned to late spermatids is distinct from background around developing sperm tails and acrosomal regions, where IFT74 localization is reported (HPA: High in elongated or late spermatids; UniProt: male germ-cell localization).
HPA tissue IHC evidence for IFT74
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Pending external verification.). 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 |
|---|---|---|---|---|
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IFT74 IHC Tips
Troubleshoot IFT74 staining in paraffin sections by assessing retrieval, compartment, tissue context and controls before interpreting chromogenic signal.
Which retrieval conditions should I try first for weak IFT74 staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08931-2). The selected tissue-IHC example used that condition before incubation with 2 μg/ml primary antibody overnight at 4°C, so keep antibody concentration and incubation consistent while assessing retrieval (datasheet A08931-2). Compare adjacent sections with and without retrieval, and inspect tissue integrity and nonspecific staining alongside the desired signal. If staining remains weak, optimize heating duration first; treat a different buffer or pH as a fallback and validate it against the EDTA condition on matched sections.
Could fixation explain weak or uneven IFT74 staining?
IFT74-specific fixation sensitivity is unknown: the selected tissue-IHC caption describes a paraffin section but does not state its fixative (datasheet A08931-2). Record fixative, fixation duration and section processing for each specimen, then compare matched material processed consistently. If staining varies across a batch, examine whether the variation follows fixation history before changing the documented EDTA pH 8.0 retrieval or 2 μg/ml primary concentration (datasheet A08931-2). Use tissue morphology and an independent staining control to judge processing quality; neither the reported tissue distribution nor protein features establish a fixation effect for this antibody.
Where should convincing IFT74 staining appear in tissue sections?
Examine the apical ciliated region in appropriate epithelia: tissue-IHC data describe membranous expression in ciliated cells of fallopian tube and respiratory epithelia (HPA: tissue IHC). Cytoplasmic staining can also occur in other tissues, while protein annotations place IFT74 along primary cilia and around the basal body or centriole (HPA: tissue IHC; UniProt Q96LB3 localisation). At chromogenic resolution, an apical line or punctum may not distinguish ciliary membrane from nearby structures, so compare serial sections with a cilia marker. Judge staining against cell identity and morphology rather than requiring every positive cell to show a sharply resolved cilium.
How should isoforms and an unknown epitope affect IHC interpretation?
The record lists 2 IFT74 isoforms, but the supplied antibody evidence does not map its epitope or establish which isoforms it detects (UniProt Q96LB3 isoforms; datasheet A08931-2). IFT74 has no annotated transmembrane segment and has reported modifications at residues 51 and 73; those facts alone do not show whether fixation or retrieval masks this antibody’s epitope (UniProt Q96LB3 topology and modified residues). Compare staining across adjacent sections using the documented EDTA pH 8.0 retrieval (datasheet A08931-2). If isoform specificity matters, obtain sequence-level antibody information or an independently validated reagent before assigning a stained compartment to one isoform.
How can IF help check a disputed chromogenic IFT74 pattern?
On the separate IF/ICC workflow, multiplex IFT74 with acetylated tubulin to identify cilia and their ciliated cells, then compare the signal with the chromogenic pattern in anatomically matched material (UniProt Q96LB3 ciliary localisation; standard IF practice). Choose a far-red IFT74 fluorophore when tissue autofluorescence obscures shorter wavelengths, and include single-channel controls to assess bleed-through (standard IF practice). Because IFT74 has no transmembrane segment, select permeabilisation that permits access to intracellular and ciliary-associated epitopes, then titrate it to preserve cilia (UniProt Q96LB3 topology; standard IF practice). IF colocalisation can support compartment assignment, but it does not establish specificity of the IHC reaction by itself.
What should I change if IFT74 DAB staining is diffuse?
First inspect a no-primary control and the tissue edges to separate detection background from antibody-associated signal (standard IHC practice). The selected paraffin-section example used 10% goat serum blocking, a peroxidase-conjugated secondary and DAB development (datasheet A08931-2). Include a peroxidase block and optimize washes and DAB development as general chromogenic-IHC steps, especially where endogenous enzyme activity or precipitate could mimic puncta (standard IHC practice). If background persists, titrate around the documented 2 μg/ml primary concentration while keeping EDTA pH 8.0 retrieval fixed, and assess whether apical ciliary staining remains distinguishable (datasheet A08931-2; UniProt Q96LB3 localisation).
How should I quantify IFT74 across differently structured sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the relevant cell population before scoring: tissue-IHC data report signal in ciliated respiratory epithelium, kidney tubules and late spermatids, among other sites (HPA: tissue IHC). For a broad cytoplasmic pattern, report the percentage of positive cells and an H-score based on intensity; for discrete apical puncta, consider positive profiles per mm² of evaluable epithelium (standard IHC practice). Normalize to the number of eligible cells or measured epithelial area, and report the fraction bearing visible cilia when that differs among samples. Apply one threshold and imaging setup across sections, excluding folds, necrosis and tissue edges before comparing groups.
When is an IFT74-positive DAB pattern likely to be artefactual?
Give greatest weight to signal in the expected cells and compartments: ciliated epithelia show a membranous pattern in tissue-IHC data, while IFT74 is annotated at cilia and basal bodies (HPA: tissue IHC; UniProt Q96LB3 localisation). Uniform nuclear staining, signal confined to damaged edges or necrosis, and deposits reproduced in a no-primary control warrant investigation as artefact (standard IHC practice). Check endogenous peroxidase activity when DAB appears independently of primary antibody, and compare staining with tissue morphology and a cilia marker on adjacent sections (standard IHC practice). A positive carcinoma example demonstrates staining under its stated conditions but does not establish that every tumour-cell signal is specific (datasheet A08931-2).
Boster reagents
Best IFT74 / Intraflagellar transport protein 74 homolog IHC Antibodies
A08931-2 has IHC images from paraffin sections of human tumors and mouse testis (catalog IHC captions); its IHC listing covers human, mouse, and rat (catalog dilution_raw).
Real IHC data
Anti-IFT74 Antibody ®
Cat # A08931-2
A08931-2 has IHC images from paraffin sections of human esophageal squamous carcinoma, lung cancer, rectum adenocarcinoma, and mouse testis (catalog IHC captions). Its listed IHC concentration is 2–5 μg/ml for human, mouse, and rat (catalog dilution_raw).
Which to pick: Choose A08931-2 for paraffin-section tissue IHC: its own captions document that preparation, while the fixative is unreported (catalog IHC captions); it is rabbit-hosted, with no clone specified (catalog host; catalog clone). No IF/ICC-validated SKU is present, so A08931-2 would require separate IF/ICC validation (catalog applications; catalog IF image alts). For cross-species IHC, A08931-2 lists human, mouse, and rat; monkey appears in overall reactivity but not in the application-specific IHC listing (catalog reactivity; catalog dilution_raw).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96LB3 (IFT74_HUMAN, Intraflagellar transport protein 74 homolog).
- Human Protein Atlas. IFT74 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. IFT74 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus, centriolar satellites and basal body. In addition localized to the cytosol..
- Human Protein Atlas. IFT74 antibody validation summary (2 antibodies).
- Loss of Ift74 Leads to Slow Photoreceptor Degeneration and Ciliogenesis Defects in Zebrafish. International journal of molecular sciences 2021 — PMC8431285.
- PubMed PMID:11683410 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IFT74 (Intraflagellar transport protein 74 homolog). Expected localisation Cilia and basal bodies (UniProt); membranous and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08931-2). Validated IHC antibody: A08931-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Elongated or late spermatids); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A08931-2); 2. Fixation: Image fixative and duration unreported (datasheet A08931-2); 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 A08931-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08931-2); 8. Primary antibody: Rabbit anti-IFT74, 2-5 μg/ml (datasheet A08931-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08931-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Cytoplasmic staining also occurs in other tissues (HPA tissue IHC); Isoform / epitope: Two isoforms; no transmembrane segment; epitope coverage unknown (UniProt); Regulation: Staining varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt Q96LB3, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the Golgi apparatus, centriolar satellites and…, Datasheet A08931-2, PMC8431285. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IFT74/immunohistochemistry-ihc-guide.