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- Table of Contents
INA / Alpha-internexin · IHC design guide
Design Immunohistochemistry for INA
Plan chromogenic INA IHC in paraffin sections using the catalog antibody’s human brain example (datasheet A03756-1). Compare neuronal cells and cerebellar processes with adrenal glandular and Leydig-cell staining when interpreting tissue results (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
INA 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 | CNS neurons and cerebellar processes; compartment unresolved (HPA tissue IHC) | |
| Staining pattern | CNS neuronal cells/processes; some adrenal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03756-1) | |
| Positive control | Cerebellum+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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03756-1) | |
| Caveat | Leydig cells also stain strongly, despite CNS enrichment (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | No annotated isoforms or cleavage; epitope map unknown (UniProt) |
Section 1
Recommended INA IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A03756-1) with two published INA staining methods (PMC3617314; PMC5039014).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A03756-1) |
| Fixation | Image fixative and duration unreported (datasheet A03756-1); 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 A03756-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03756-1) |
| Primary antibody | Rabbit anti-INA, 2-5μg/ml (datasheet A03756-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03756-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03756-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | INA-positive staining in processes in granular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Distinct expression in the CNS and in a subset of adrenal gland cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA, pH 8.0 (datasheet A03756-1); citrate, pH 6.0, is a published alternative (PMC3617314 methods).
Section 2
What Is the Expected INA Staining Pattern?
INA is a neuronal intermediate filament enriched in the adult CNS (UniProt Q16352). In tissue IHC, expect staining in cerebellar granular-layer processes and neurons in the caudate, cerebral cortex, and hippocampus (HPA tissue IHC: Enhanced). HPA ICC-IF places INA mainly in intermediate filaments, with additional cytosolic signal (HPA subcellular: approved). INA has no transmembrane segment (UniProt Q16352 topology).
| Strong staining follows processes in the cerebellar granular layer. | This matches HPA's High cerebellar process signal (HPA tissue IHC). Assess the process pattern alongside tissue structure; a positive field alone cannot establish antibody specificity (general IHC practice). |
| Neuronal cells stain in the caudate, cerebral cortex, or hippocampus. | These are reported at Medium intensity (HPA tissue IHC). Cell-associated staining is compatible with INA's neuronal filament role (UniProt Q16352 function); intensity can vary across regions. |
| Predominantly nuclear staining replaces the expected filament-associated pattern. | HPA places INA mainly in intermediate filaments, with additional cytosolic localization in ICC-IF (HPA subcellular: approved). Review nuclear signal as possible nonspecific staining; ICC-IF localization is supporting evidence, not a direct FFPE compartment validation. |
| Strong staining appears in an unexpected cell population or across a nominally negative tissue. | Cross-reactivity or endogenous chromogen-generating activity may explain unexpected signal (general IHC practice). Check the specific cell type: HPA reports High Leydig-cell staining, so testis is not a blanket negative (HPA tissue IHC). |
| The known-positive region is blank, or low-level color covers most of the section. | A blank cerebellar granular layer conflicts with its reported High signal (HPA tissue IHC). Widespread color obscuring cell structure favors background; inspect controls and detection conditions before interpreting either result (general IHC practice). |
💡Expected INA appearanceCall positive when cerebellar granular-layer processes show High staining or the listed neuronal populations show Medium staining (HPA tissue IHC); diffuse nuclear color or indiscriminate tissue-wide color warrants investigation (HPA subcellular: approved; general IHC practice).
| Tissue and cell selection | HPA reports High cerebellar process and Leydig-cell staining, Medium staining in listed brain neurons and adrenal glandular cells, and no detected signal in several specified non-neural cell populations (HPA tissue IHC). Select controls by both tissue and cell type. |
| Compartment evidence | Intermediate filaments are the approved main ICC-IF location; cytosol is additional (HPA subcellular). UniProt provides no subcellular annotation (UniProt Q16352). Use the ICC-IF result to guide review of IHC morphology without treating it as an IHC compartment measurement. |
| Antibody validation | Two listed antibodies, HPA008057 and CAB002059, have Enhanced IHC status; HPA007572 has Approved ICC status with no listed IHC status (HPA antibodies). These statuses are application-specific and do not establish the performance of an unlisted catalog antibody. |
| INA form and modification | UniProt lists one chain spanning residues 1–499, no signal peptide or propeptide, no listed isoforms, and several modified residues (UniProt Q16352). The payload gives no antibody epitope, so it does not support a prediction that any modification changes staining. |
| Antigen retrieval and fixation | The supplied HPA and UniProt records give no target-specific retrieval condition or fixation sensitivity. If a positive control fails, optimize retrieval against the antibody's validated IHC instructions while retaining matched controls (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in cerebellar granular-layer processes | Possible staining-run failure; this region is reported High (HPA tissue IHC). | Check the positive-control section, antibody and detection steps, and retrieval conditions using the antibody's IHC instructions (general IHC practice). |
| Diffuse color obscures cellular detail | Excess background can arise from concentrated primary antibody, inadequate blocking, or detection reagent activity (general IHC practice). | Compare a no-primary control, then adjust blocking, washing, antibody concentration, or chromogen development as indicated (general IHC practice). |
| Nuclei dominate the signal | This disagrees with the filament and cytosol locations reported by ICC-IF (HPA subcellular: approved); nonspecific staining is possible. | Check the counterstain and no-primary control; confirm whether process-associated staining remains visible in a known-positive region (general IHC practice). |
| Unexpected cells stain strongly | Cross-reactivity or endogenous peroxidase activity may contribute in chromogenic IHC (general IHC practice). Leydig cells are a reported High exception to an adult-CNS-only expectation (HPA tissue IHC; UniProt Q16352 tissue specificity). | Identify the exact cell population, compare HPA tissue data, and inspect no-primary and peroxidase-block controls (general IHC practice). |
| A presumed negative control stains | A whole-tissue negative assumption may be too broad: HPA levels apply to named cells, such as undetected adipocytes in adipose tissue and undetected glandular cells in appendix (HPA tissue IHC). | Score the annotated cell population rather than the entire section; investigate signal elsewhere with matched controls (general IHC practice). |
| IF/ICC: what pattern should I expect? | HPA reports mainly intermediate-filament localization, plus cytosol, in ICC-IF (HPA subcellular: approved). | Assess filament-associated and cytosolic signal against the IF/ICC guide's controls; the IHC tissue pattern and IHC antibody statuses do not establish an IF/ICC protocol (HPA tissue IHC; HPA antibodies). |
Experiment evidence
IHC & IF images showing INA staining
A03756-1 · IHCIHC analysis of Alpha Internexin/INA using anti-Alpha Internexin/INA antibody (A03756-1). Alpha Internexin/INA was detected in a paraffin-em
A03756-1 · IHCIHC analysis of Alpha Internexin/INA using anti-Alpha Internexin/INA antibody (A03756-1). Alpha Internexin/INA was detected in a paraffin-em
A03756-1 · IFIF analysis of Alpha Internexin/INA using anti-Alpha Internexin/INA antibody (A03756-1). Alpha Internexin/INA was detected in an immunocytoc
PB10003 · IHCIHC analysis of Alpha Internexin using anti-Alpha Internexin antibody (PB10003). Alpha Internexin was detected in a paraffin-embedded sectio
PB10003 · IHCIHC analysis of Alpha Internexin using anti-Alpha Internexin antibody (PB10003). Alpha Internexin was detected in a paraffin-embedded sectio
PB10003 · IFIF analysis of Alpha Internexin using anti-Alpha Internexin antibody (PB10003). Alpha Internexin was detected in an immunocytochemical secti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000148798-INA/tissue/cerebral+cortex#imid_668928
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000148798-INA/tissue/adrenal+gland#imid_668912
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000148798-INA/tissue/colon#imid_668854
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000148798-INA/tissue/liver#imid_668863
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA007572; green = INA, blue = nucleus, red = microtubules.
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA007572; green = INA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for INA IHC & IF
🧪Run cerebellum first and look for staining in processes of the granular layer (HPA: High in processes in granular layer). Use adipose tissue as the negative, focusing on adipocytes (HPA: Not detected in adipocytes); on the cerebellum slide, nuclei should lack the filament-like staining seen in those processes (HPA: intermediate filaments; High in processes in granular layer).
Positive control tissue: Cerebellum (Processes in granular layer, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show INA in SH-SY5Y, SiHa, U2OS, with annotated localisation: Intermediate filaments (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched nonimmune rabbit IgG control alongside INA knockout tissue, if available, as a biological negative (caption: rabbit primary antibody; standard IHC practice). For the caption’s biotin-based DAB detection, quench endogenous peroxidase and assess endogenous biotin background on cerebellum sections (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A03756-1 paraffin-section caption does not state the fixative (caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 is documented for the mouse brain example, but whether INA staining depends on that retrieval condition is unreported (caption: EDTA retrieval, pH 8.0). HPA shows ICC-IF localization to intermediate filaments, with additional cytosolic signal, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; assess background from the caption’s biotin-based DAB detection in the chosen tissue (HPA: subcellular localization; caption: SABC and DAB; standard IHC practice).
HPA tissue IHC evidence for INA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 | Processes in granular layer | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced INA IHC Tips
Troubleshoot INA staining in paraffin sections by checking retrieval, tissue processing, filament localisation and controls before scoring the chromogenic signal.
What retrieval should I start with if INA staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A03756-1). The selected mouse brain example used that retrieval before 2 μg/ml primary antibody overnight at 4°C and DAB detection (datasheet A03756-1). If staining remains weak, compare a small range of heating durations on adjacent sections while keeping the antibody concentration, development time and imaging conditions constant (standard IHC practice). Include a known positive neural section and a primary-omission control; excessive heating can damage morphology and make apparent signal gains difficult to interpret (standard IHC practice).
Could fixation be causing weak or patchy INA staining?
Target-specific sensitivity of INA to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet A03756-1). Record the fixative, fixation duration and processing history for each specimen before attributing differences to antigen loss (standard IHC practice). Compare sections processed together, beginning with the documented EDTA pH 8.0 retrieval and 2 μg/ml primary antibody conditions (datasheet A03756-1). If signal varies by batch, test adjacent sections with controlled retrieval durations and a positive control, then judge staining alongside tissue morphology rather than assuming a particular fixation effect on INA (standard IHC practice).
Where should I expect INA signal, and when is localisation suspicious?
Look for filament-associated staining in neuronal cells and processes: INA is a class-IV neuronal intermediate filament, and its approved cellular location is mainly intermediate filaments with additional cytosolic localisation (UniProt Q16352 function; HPA subcellular). Cerebellar granular-layer processes show high staining, while caudate, cortical and hippocampal neuronal cells show medium staining in the HPA tissue profile (HPA tissue IHC). Examine those structures at matched magnification before scoring a diffuse DAB haze as positive (standard IHC practice). Predominantly nuclear or sharply extracellular deposits do not fit the reported cellular locations; compare them with a primary-omission section and nearby intact tissue (HPA subcellular; standard IHC practice).
Can INA isoforms or modifications explain different staining patterns?
The supplied INA record lists 0 isoforms, one chain spanning residues 1–499, and an intermediate-filament rod at residues 94–407 (UniProt Q16352). It also lists phosphorylation sites, including serines 72 and 219, plus acetylation at lysine 290 (UniProt Q16352). Because the selected antibody's epitope is not specified in the supplied caption, these modifications cannot be assigned as the cause of a staining difference (datasheet A03756-1; UniProt Q16352). Check the antibody's documented immunogen or epitope, if available, and compare the same retrieval and detection conditions across adjacent sections before proposing an epitope-dependent effect (standard IHC practice).
How can I check an INA IHC pattern with multiplex immunofluorescence?
Use IF as a separate validation experiment and pair INA with a marker for the expected neuronal cell type or processes, selected and validated for the specimen (UniProt Q16352 function; standard IF practice). Choose spectrally separated fluorophores and include unstained tissue to identify autofluorescence before interpreting overlap (standard IF practice). INA has no transmembrane segment and is mainly associated with intracellular intermediate filaments, so optimise permeabilisation for an intracellular epitope while preserving process morphology (UniProt Q16352 topology; HPA subcellular; standard IF practice). The EDTA pH 8.0 retrieval and 2 μg/ml antibody condition document the selected chromogenic tissue example, not an IF protocol (datasheet A03756-1).
How do I reduce diffuse brown background without losing INA processes?
First inspect a primary-omission control and a known positive neural section developed for the same DAB time (standard IHC practice; HPA tissue IHC). The selected example used 10% goat serum blocking, 2 μg/ml rabbit primary overnight at 4°C, and a biotinylated secondary with SABC and DAB (datasheet A03756-1). If background is widespread, adjust primary concentration, washing and development time one variable at a time; include peroxidase blocking as a general chromogenic IHC step (standard IHC practice). Check whether diffuse signal follows tissue edges or damaged areas rather than filament-rich neuronal structures, and document any change in positive-control staining (HPA subcellular; standard IHC practice).
How should I quantify INA staining when much of the signal lies in processes? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region of interest before scoring and keep section thickness, DAB development, illumination and image thresholds consistent across specimens (standard IHC practice). For neuronal cell bodies, report an H-score or percentage of positive cells against all evaluable neuronal cells; for process-rich regions, measure stained area or optical density per mm² of intact tissue (standard IHC practice). Cerebellar granular-layer processes are a documented high-signal reference, whereas caudate, cortical and hippocampal neuronal cells have medium staining in the supplied profile (HPA tissue IHC). Exclude folds, necrosis and section edges consistently, and report cell-based and process-based measurements separately (standard IHC practice).
How can I distinguish genuine INA positivity from staining artefact?
Treat filament-associated signal in neuronal cells or processes as more plausible than isolated nuclear, extracellular or edge-only DAB deposits (UniProt Q16352 function; HPA subcellular; standard IHC practice). The supplied tissue profile reports high staining in cerebellar granular-layer processes and medium staining in neuronal cells of the caudate, cerebral cortex and hippocampus (HPA tissue IHC). Compare suspect fields with intact adjacent tissue and a primary-omission control to identify section-edge effects, necrotic deposits and endogenous enzyme signal (standard IHC practice). The selected EDTA pH 8.0 mouse brain example establishes a tested chromogenic workflow, but a positive call still requires the expected cellular pattern and clean controls (datasheet A03756-1; standard IHC practice).
Boster reagents
Best INA / Alpha-internexin IHC Antibodies
Two IHC-validated anti-INA antibodies have paraffin-section images from mouse brain and human glioma, plus IF images from cells and brain sections (catalog image captions). Both list human, mouse and rat reactivity (catalog applications/reactivity).
Real IHC data
Anti-alpha Internexin/INA Antibody ®
Cat # A03756-1
Real IHC data
Anti-Alpha Internexin/INA Antibody ®
Cat # PB10003
A03756-1 will render with its mouse brain paraffin-section IHC image; its catalog also shows IHC in rat and human brain and IF in SiHa cells and rat brain (A03756-1 image captions). PB10003 will render with its human glioma paraffin-section IHC image; its catalog also shows IHC in mouse and rat brain and IF in A549 cells and mouse and rat brain (PB10003 image captions).
Which to pick: For human tissue IHC, start with PB10003 because its displayed IHC image uses a paraffin section of human glioma; for mouse brain IHC, A03756-1 has a matching displayed image (respective IHC image captions). For IF/ICC, choose A03756-1 for SiHa cells or PB10003 for A549 cells, matching their IF images (respective IF image captions). Both are rabbit antibodies listed for IHC, IF and ICC in human, mouse and rat, so either is a cross-species candidate (catalog applications/reactivity); their paraffin-section captions do not report the fixative (respective IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16352 (AINX_HUMAN, Alpha-internexin).
- Human Protein Atlas. INA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. INA subcellular location (ICC-IF): Mainly localized to the intermediate filaments. In addition localized to the cytosol..
- Human Protein Atlas. INA antibody validation summary (3 antibodies).
- Alpha internexin expression related with molecular characteristics in adult glioblastoma and oligodendroglioma. Journal of Korean medical science 2013 — PMC3617314.
- The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms. Cancer 2012 — PMC3312048.
- Spatial patterns of FUS-immunoreactive neuronal cytoplasmic inclusions (NCI) in neuronal intermediate filament inclusion disease (NIFID). Journal of neural transmission (Vienna, Austria : 1996) 2011 — PMC3199334.
- A truncating SOD1 mutation, p.Gly141X, is associated with clinical and pathologic heterogeneity, including frontotemporal lobar degeneration. Acta neuropathologica 2015 — PMC5039014.
- PubMed PMID:7769995 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for INA (Alpha-internexin). Expected localisation CNS neurons and cerebellar processes; compartment unresolved (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03756-1). Validated IHC antibody: A03756-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Processes in granular layer); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A03756-1); 2. Fixation: Image fixative and duration unreported (datasheet A03756-1); 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 A03756-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03756-1); 8. Primary antibody: Rabbit anti-INA, 2-5μg/ml (datasheet A03756-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03756-1). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03756-1); Caveat: Leydig cells also stain strongly, despite CNS enrichment (HPA tissue IHC); Isoform / epitope: No annotated isoforms or cleavage; epitope map unknown (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt Q16352, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the intermediate filaments. In addition…, Datasheet A03756-1, PMC3617314, PMC5039014. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/INA/immunohistochemistry-ihc-guide.