IARS2 / Isoleucine--tRNA ligase, mitochondrial · IHC design guide

Design Immunohistochemistry for IARS2

This guide uses the granular cytoplasmic tissue pattern and high staining in glandular cells and kidney tubules to plan IARS2 paraffin IHC (HPA tissue IHC). It covers controls and scoring alongside the catalog antibody's 2–5 μg/ml IHC dilution (datasheet A09580-2).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for IARS2 (IHC for IARS2): expected localisation Observed granular cytoplasm (HPA tissue IHC); mitochondrial matrix expected (UniProt), antibody A09580-2, validated IHC image, and IHC protocol steps — Printable **IARS2** IHC protocol sheet — expected localisation Observed granular cytoplasm (HPA tissue IHC); mitochondrial matrix expected (UniProt), antibody A09580-2, controls and protocol steps. Open the full IARS2 IHC guide →

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IARS2 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	Observed granular cytoplasm (HPA tissue IHC); mitochondrial matrix expected (UniProt)
	Staining pattern	Granular cytoplasm; high in glandular and kidney tubule cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A09580-2)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections (standard IHC practice; not target-specific)
	Caveat	Staining and RNA expression show medium consistency (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	No isoforms; mature chain spans residues 49–1012 (UniProt)

Section 1

Recommended IARS2 IHC & IF Protocols

The catalog antibody has an IHC-P protocol (datasheet: A09580-2). Two published IARS2 IHC methods provide additional tissue-section workflows (PMC11234349; PMC7377047).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet A09580-2)
Fixation	Image fixative and duration unreported (datasheet A09580-2); 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 A09580-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A09580-2)
Primary antibody	Rabbit anti-IARS2, 2-5 μg/ml (datasheet A09580-2)
Primary incubation	Overnight at 4 °C (datasheet A09580-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A09580-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	IARS2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in all tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A09580-2); the published methods mention retrieval without specifying a buffer (PMC11234349; PMC7377047).

Section 2

What Is the Expected IARS2 Staining Pattern?

IARS2 is a mitochondrial matrix protein with no transmembrane segment (UniProt Q9NSE4: location and topology). In paraffin sections, expect granular cytoplasmic staining in cells across tissues, including glandular cells and kidney tubule cells (HPA: tissue IHC). HPA rates its tissue IHC reliability Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA: tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic signal in colon glandular cells or kidney tubule cells (HPA: High in both).	This matches the reported tissue pattern and the expected mitochondrial compartment (HPA: tissue IHC; UniProt Q9NSE4: mitochondrial matrix). Judge the granules against nearby background before scoring intensity (general IHC practice).
Predominantly nuclear or sharply membrane-bound staining, without granular cytoplasmic signal.	This conflicts with the reported localisation (HPA: granular cytoplasmic tissue staining; UniProt Q9NSE4: mitochondrial matrix, no transmembrane segment). Treat it as a possible artefact and check the detection controls before assigning it to IARS2 (general IHC practice).
Strong signal restricted to an unexpected cell population while expected cells are unstained.	Compare the cell identity with the HPA observations; for example, kidney tubule cells are High (HPA: tissue IHC). Cross-reactivity or endogenous chromogenic detection activity are possible explanations, but this pattern alone cannot distinguish them (general IHC practice).
Uniform colour across cells and surrounding tissue, with no resolvable cytoplasmic granules.	Diffuse background obscures the reported granular pattern (HPA: tissue IHC). Review blocking, antibody concentration and wash conditions, and compare a reagent-omission control (general IHC practice). Do not score uniform colour as specific IARS2 staining.
No visible signal in colon glandular cells or kidney tubule cells.	Both populations are reported High, so a blank slide warrants a workflow check (HPA: tissue IHC). Confirm the tissue identity and inspect retrieval, antibody and chromogenic detection controls (general IHC practice). A single blank section does not establish absent IARS2 expression.

💡Expected IARS2 appearanceCall positive a discernible granular cytoplasmic signal in expected cells, especially High-staining glandular or kidney tubule cells; isolated nuclear, membrane-bound or uniform background colour is suspect (HPA: tissue IHC; UniProt Q9NSE4: mitochondrial matrix).

How each factor affects the staining
Cell population and tissue	HPA reports granular cytoplasmic staining across tissues, with High levels in several glandular populations, kidney tubule cells and lymph-node non-germinal-center cells (HPA: tissue IHC). Its listed Low populations are weak comparators, not validated negative controls (HPA: tissue IHC).
Compartment and topology	A mitochondrial matrix protein without a transmembrane segment supports a granular cytoplasmic interpretation (UniProt Q9NSE4: location and topology; HPA: tissue IHC). The record does not identify an antibody epitope or establish how antigen retrieval affects its detection.
Evidence strength	HPA labels tissue IHC Enhanced but describes medium consistency with RNA expression (HPA: tissue IHC). Its listed antibodies have IHC ratings of Enhanced for HPA024212 and HPA024594, and Supported for HPA024596 (HPA: antibody validation); these ratings do not validate every staining pattern.
IF/ICC Q&A: what pattern should be checked?	Look for mitochondrial localisation: HPA describes IARS2 as mainly mitochondrial in ICC-IF, with an enhanced main-location rating (HPA: subcellular). This supports a compartment cross-check; ICC-IF images do not supply a paraffin-section protocol or prove that every IHC granule is mitochondrial.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
High-staining reference tissue is blank (HPA: colon glandular cells or kidney tubules).	Tissue selection or a general retrieval, antibody or detection failure may explain the result (general IHC practice).	Verify the cell population, run a known-positive section alongside the sample, and check retrieval and detection steps (general IHC practice). No IARS2-specific fixation sensitivity is established by the supplied sources.
Signal is strong but predominantly nuclear or outlines cell membranes.	The compartment conflicts with mitochondrial matrix localisation and granular cytoplasmic IHC (UniProt Q9NSE4; HPA: tissue IHC).	Review morphology and controls; compare staining with expected cells before treating the signal as specific (general IHC practice).
Unexpected cells stain while expected cells do not.	Cross-reactivity or endogenous detection activity is possible; the image alone cannot identify which (general IHC practice).	Check a reagent-omission control and an HPA High cell population, then reassess cell identity and localisation (general IHC practice; HPA: tissue IHC).
The whole section has diffuse colour that hides granules.	Excess background from blocking, antibody concentration, washing or detection is possible (general IHC practice).	Inspect controls and adjust one workflow variable at a time until granular cytoplasmic signal separates from background (general IHC practice; HPA: tissue IHC).
A listed Low population stains faintly or inconsistently.	HPA reports Low staining in several populations, including skeletal-muscle myocytes and smooth-muscle cells (HPA: tissue IHC).	Avoid using a Low population as a definitive negative control; compare it with a reported High population on the same run (HPA: tissue IHC; general IHC practice).
ICC-IF appears mitochondrial, but IHC is diffuse.	HPA reports mitochondrial ICC-IF localisation and granular cytoplasmic tissue IHC; the discordance remains unresolved by these observations alone (HPA: subcellular and tissue IHC).	Interpret each preparation with its own controls and review IHC background and cell morphology (general IHC practice). Do not substitute an ICC-IF result for paraffin-section validation.

Source: UniProt Q9NSE4 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing IARS2 staining

IHC analysis of IARS2 using anti-IARS2 antibody (A09580-2). IARS2 was detected in a paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-IARS2 Antibody (A09580-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A09580-2 · IHCIHC analysis of IARS2 using anti-IARS2 antibody (A09580-2). IARS2 was detected in a paraffin-embedded section of human esophageal squamous c

IHC analysis of IARS2 using anti-IARS2 antibody (A09580-2). IARS2 was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-IARS2 Antibody (A09580-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A09580-2 · IHCIHC analysis of IARS2 using anti-IARS2 antibody (A09580-2). IARS2 was detected in a paraffin-embedded section of human placenta tissue. Heat

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000067704-IARS2/tissue/cerebral+cortex#imid_6516440

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000067704-IARS2/tissue/colon#imid_6515006

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000067704-IARS2/tissue/liver#imid_6515007

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000067704-IARS2/tissue/kidney#imid_6515000

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA024594; green = IARS2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for IARS2 IHC & IF

🧪Run adrenal gland first: its glandular cells should stain strongly (HPA: High in adrenal gland glandular cells). HPA detects IARS2 in all 45 scored tissues, so there is no HPA negative tissue; use no-primary and isotype controls, and expect nuclei within stained glandular cells to lack mitochondrial signal rather than treating other cell types as proven negatives (HPA: no negative rows; UniProt Q9NSE4: mitochondrial matrix).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: IARS2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IARS2 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a secondary-only slide, a control using rabbit IgG matched to the primary antibody’s clonality where known, and IARS2-knockout material as a biological negative (caption: rabbit anti-IARS2 primary). For chromogenic detection, block endogenous peroxidase and check for tissue background before interpreting DAB staining (caption: peroxidase secondary and DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09580-2 paraffin-section caption does not state the fixative (selected tissue-IHC caption). The caption reports heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required under other conditions (selected tissue-IHC caption). HPA shows mitochondrial ICC-IF staining in A-431, U-251MG and U2OS cells; the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC, and DAB background should be assessed in the chosen tissue (HPA: subcellular; selected tissue-IHC caption: DAB).

HPA tissue IHC evidence for IARS2

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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Endometrium	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: IARS2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced IARS2 IHC Tips

Use the catalog antibody’s paraffin-section IHC conditions as the starting point, then judge staining against IARS2’s mitochondrial localisation and tissue expression.

Which retrieval conditions should I start with for IARS2 paraffin-section IHC?

Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A09580-2). The selected tissue image used that retrieval before 2 μg/ml primary antibody overnight at 4°C, providing a tested starting combination for this SKU (datasheet A09580-2). If staining is weak, vary heating time on matched sections while keeping EDTA pH 8.0 constant; excessive heating can damage morphology (standard IHC practice). Compare each condition with a matched section lacking primary antibody, and assess whether cytoplasmic granules become clearer without increased diffuse staining (standard IHC practice; HPA: granular cytoplasmic expression).

How should I troubleshoot fixation-related loss of IARS2 staining?

The selected caption identifies a paraffin-embedded section but does not state its fixative, so IARS2-specific fixation sensitivity is unknown (datasheet A09580-2 caption). Record the fixative and fixation duration for each specimen, then compare matched sections processed with the same EDTA pH 8.0 retrieval and antibody incubation (standard IHC practice; datasheet A09580-2). Assess preserved cellular detail alongside staining, because damaged morphology makes a granular cytoplasmic pattern difficult to interpret (standard IHC practice; HPA: granular cytoplasmic expression). Do not attribute weak staining to a particular fixative without a controlled comparison on otherwise similar specimens (standard IHC practice).

Where should convincing IARS2 staining appear in tissue sections?

Expect a granular cytoplasmic signal consistent with mitochondrial localisation, while recognising that chromogenic IHC cannot resolve individual mitochondrial membranes (HPA: granular cytoplasmic expression; UniProt Q9NSE4: mitochondrial matrix; standard IHC practice). IARS2 has no annotated transmembrane segment, so a crisp cell-surface rim would conflict with its recorded topology (UniProt Q9NSE4 topology). Examine positive cells at high magnification and compare their cytoplasmic pattern with nearby negative-control sections (standard IHC practice). HPA reports high staining in kidney tubular cells and several glandular cell populations, which can serve as tissue-pattern references without establishing specificity for this antibody (HPA: tissue IHC).

Could epitope processing or modification explain inconsistent IARS2 IHC?

UniProt annotates a processed IARS2 chain spanning residues 49–1012, while the supplied record provides no antibody epitope position (UniProt Q9NSE4 processing; supplied antibody evidence). It lists 0 annotated isoforms and several modified residues, including acetylation and succinylation, but those annotations do not establish that modification changes this antibody’s staining (UniProt Q9NSE4 isoforms and modified residues). If samples stain differently, compare matched sections using the same EDTA pH 8.0 retrieval before assigning the difference to epitope accessibility (datasheet A09580-2; standard IHC practice). Seek epitope-mapping or independent validation evidence before interpreting a staining difference as isoform- or modification-specific (standard IHC practice).

How can I check an IHC pattern with multiplex IF?

For the separate IF/ICC workflow, pair IARS2 with a mitochondrial marker and a marker for the expected cell type, such as an epithelial marker when assessing glandular cells (UniProt Q9NSE4: mitochondrial matrix; HPA: glandular-cell staining; standard IF practice). Place the weaker signal in a bright, spectrally distinct channel and choose fluorophores after checking tissue autofluorescence in an unstained control (standard IF practice). Because the expected epitope is inside mitochondria, optimise detergent permeabilisation, starting with 0.1% Triton X-100, to permit access to a matrix protein (UniProt Q9NSE4: mitochondrial matrix; standard IF practice). Confirm mitochondrial overlap and cell identity separately; this IHC caption supplies no IF/ICC validation for the catalog antibody (datasheet A09580-2 caption).

What should I change if IARS2 DAB staining is diffuse?

First compare the section with a matched no-primary control to identify signal from detection reagents or endogenous peroxidase (standard IHC practice). The selected IHC image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (datasheet A09580-2). If background persists, check the wash steps, titrate primary antibody around that documented concentration, and use a peroxidase block before DAB development (standard IHC practice; datasheet A09580-2). Judge improvement by retention of granular cytoplasmic staining while diffuse deposits and staining in the no-primary control decrease (HPA: granular cytoplasmic expression; standard IHC practice).

How should I quantify IARS2 chromogenic staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population and viable tissue area before examining staining intensity, and apply the same inclusion rules to every section (standard IHC practice). For cell-based scoring, report the percentage of positive cells and an H-score from 0–300 using intensity categories 0–3 (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when cell counts are impractical (standard IHC practice). Keep section thickness, retrieval, antibody concentration, DAB development, and image settings consistent, and exclude necrosis and edges from comparisons (standard IHC practice).

How do I distinguish true IARS2 staining from IHC artefact?

Give greatest weight to granular cytoplasmic staining in intact cells, which matches the reported tissue pattern and mitochondrial location of IARS2 (HPA: granular cytoplasmic expression; UniProt Q9NSE4: mitochondrial matrix). Diffuse nuclear staining or a cell-surface outline conflicts with that localisation and warrants review of controls and morphology (UniProt Q9NSE4 localisation and topology; standard IHC practice). Check whether apparent positives cluster at section edges or in necrotic areas, where staining can be misleading (standard IHC practice). A no-primary control helps reveal endogenous enzyme or detection-related colour, while HPA’s enhanced tissue reliability has only medium staining–RNA consistency and does not validate every individual section (standard IHC practice; HPA: tissue IHC reliability).

Boster reagents

Best IARS2 / Isoleucine--tRNA ligase, mitochondrial IHC Antibodies

A09580-2 has anti-IARS2 IHC images from paraffin-embedded human carcinoma and placenta samples and mouse kidney (catalog: IHC captions). Human, mouse, and rat reactivity is listed; IF/ICC data are absent (catalog: applications/reactivity/images).

Real IHC data

Anti-IARS2 Antibody ®

Cat # A09580-2

View product →

A09580-2 is listed for IHC in human, mouse, and rat samples (catalog: applications/reactivity). Its IHC captions show paraffin-embedded human esophageal squamous carcinoma, placenta, and rectum adenocarcinoma, plus mouse kidney (catalog: A09580-2 IHC captions).

Which to pick: For paraffin-section tissue IHC, choose A09580-2; its captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody, while the fixative is unreported (catalog: A09580-2 IHC captions). No IF/ICC option is supported, and clone type is unreported (catalog: A09580-2 applications/IF images/clone). For work across species, A09580-2 lists human, mouse, and rat reactivity, with IHC images provided for human and mouse samples (catalog: A09580-2 reactivity/IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q9NSE4 (SYIM_HUMAN, Isoleucine--tRNA ligase, mitochondrial).
Human Protein Atlas. IARS2 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. IARS2 subcellular location (ICC-IF): Mainly localized to the mitochondria..
Human Protein Atlas. IARS2 antibody validation summary (3 antibodies).
Unveiling isoleucyl-tRNA synthetase 2 as a novel driver of breast cancer via β-catenin pathway activation. Journal of cell communication and signaling 2026 — PMC13356825.
Mitochondrial protein isoleucyl-tRNA synthetase 2 in tumor cells as a potential therapeutic target for cervical cancer. CytoJournal 2024 — PMC11234349.
RNAi-mediated IARS2 knockdown inhibits proliferation and promotes apoptosis in human melanoma A375 cells. Oncology letters 2020 — PMC7377047.
Lentivirus-induced knockdown of IARS2 expression inhibits the proliferation and promotes the apoptosis of human osteosarcoma cells. Oncology letters 2022 — PMC9219035.
PubMed PMID:16710414 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.