AARS2 / Alanine--tRNA ligase, mitochondrial · IHC design guide

Design Immunohistochemistry for AARS2

Plan AARS2 paraffin-section IHC using the cytoplasmic tissue pattern as a reference (HPA tissue IHC). The catalog antibody’s colon-section example documents primary-antibody incubation and DAB detection (datasheet A10160-2).

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

Immunohistochemistry protocol sheet for AARS2 (IHC for AARS2): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt), antibody A10160-2, validated IHC image, and IHC protocol steps — Printable **AARS2** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt), antibody A10160-2, controls and protocol steps. Open the full AARS2 IHC guide →

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AARS2 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	Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial protein (UniProt)
	Staining pattern	Glandular cells show cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A10160-2)
	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	Colon endothelial cells can stain strongly (HPA tissue IHC)
	Regulation	Expression regulation not annotated (UniProt)
	Isoform / epitope	No isoforms annotated; mature chain starts at aa 24 (UniProt)

Section 1

Recommended AARS2 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with one published AARS2 IHC protocol for human colon adenocarcinoma specimens (PMC12982081).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A10160-2)
Fixation	Image fixative and duration unreported (datasheet A10160-2); 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 A10160-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A10160-2)
Primary antibody	Rabbit anti-AARS2, 1:50 recommended; image 1:200 (datasheet A10160-2)
Primary incubation	Overnight at 4 °C (datasheet A10160-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A10160-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AARS2-positive staining in glandular cells of adrenal gland (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 retrieval for paraffin sections (datasheet A10160-2); the published protocol does not specify retrieval (PMC12982081).

Section 2

What Is the Expected AARS2 Staining Pattern?

AARS2 is mitochondrial and has no transmembrane segment (UniProt Q5JTZ9 localisation and topology). In paraffin sections, expect cytoplasmic staining in glandular, respiratory epithelial and other HPA-positive cells (HPA: general cytoplasmic expression; listed High cells). HPA rates the tissue IHC pattern Approved, with medium consistency between staining and RNA expression (HPA: tissue IHC reliability). Interpret intensity alongside cell identity and compartment.

What am I looking at on my slide?
Cytoplasmic staining in adrenal or duodenal glandular cells, with little nuclear staining.	This fits the expected compartment and a documented High cell population (UniProt Q5JTZ9: mitochondrion; HPA: High in these glandular cells). Chromogenic IHC may show cytoplasmic signal without resolving individual mitochondria (general IHC practice).
Signal confined mainly to nuclei, cell borders or extracellular material.	Those distributions conflict with the mitochondrial assignment (UniProt Q5JTZ9; HPA: mitochondrial ICC-IF). Treat them as suspect; compare with a known-positive section and control staining before assigning AARS2 (general IHC practice).
Strong staining in a cell population absent from the selected HPA High entries.	Check cell identity and assay controls before calling cross-reactivity: HPA reports low tissue specificity, and its supplied High list is not an exhaustive negative map (HPA: tissue RNA specificity and listed IHC entries). Endogenous detection activity can also mimic chromogen signal (general IHC practice).
Diffuse colour covers cells, stroma and the blank area around tissue.	A field-wide deposit is less persuasive than cell-associated cytoplasmic staining (HPA: general cytoplasmic expression). Assess nonspecific detection, wash quality and chromogen development with appropriate controls (general IHC practice).
No staining in a section containing a documented High cell population.	First verify that the expected cells are present and the control run worked (HPA: listed High cells; general IHC practice). A negative slide alone cannot establish absent AARS2; HPA's IHC rating is Approved with medium staining–RNA consistency (HPA: reliability).

💡Expected AARS2 appearanceCall a result positive when the documented High cells show clear cytoplasmic signal consistent with mitochondrial AARS2 (HPA: tissue IHC; UniProt Q5JTZ9); isolated nuclear or field-wide colour is suspect (general IHC practice).

How each factor affects the staining
Compartment	UniProt places AARS2 in mitochondria, and HPA ICC-IF shows mitochondrial localisation (UniProt Q5JTZ9; HPA: subcellular ICC-IF). Judge paraffin IHC at the cytoplasmic level when organelles cannot be resolved (general IHC practice).
Choice of reference cells	Adrenal, appendix, breast, cervix and duodenum glandular cells; bronchial respiratory epithelial cells; cerebellar granular-layer cells; and colonic endothelial cells are listed High (HPA: tissue IHC). Adipocytes are listed Low (HPA: adipose tissue).
Strength of antibody evidence	HPA035636 has Approved IHC status; the supplied record gives no IHC status for HPA061396 (HPA: antibody validation). Both have Enhanced ICC status (HPA: antibody validation). Do not transfer an ICC validation rating to paraffin IHC.
Target structure and epitope information	UniProt reports no transmembrane segment and a chain spanning residues 24–985 (UniProt Q5JTZ9: topology and processing). The supplied sources do not map the IHC antibody epitope or establish target-specific retrieval or fixation sensitivity.
Chromogenic detection	Endogenous enzyme activity, nonspecific secondary binding and excessive chromogen development can create misleading colour (general IHC practice). Use assay-matched negative controls to distinguish detection background from the HPA cytoplasmic pattern.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A documented High tissue is blank.	The expected cell population may be absent from the section, or the staining run may have failed (HPA: High cell listings; general IHC practice).	Confirm cell identity and review the run's positive control; then check retrieval, antibody dilution and detection using the validated IHC procedure (general IHC practice). No AARS2-specific retrieval condition is supplied.
Colour is strongest outside cells.	Background deposition or nonspecific detection can obscure the expected cytoplasmic pattern (HPA: general cytoplasmic expression; general IHC practice).	Inspect an assay-matched negative control, washing and chromogen development; reassess whether staining remains cell-associated after background is controlled (general IHC practice).
Only nuclei or cell borders stain.	The observed compartment disagrees with the mitochondrial localisation (UniProt Q5JTZ9; HPA: subcellular ICC-IF).	Compare compartment placement in a known-positive section and check control staining before scoring the result as AARS2 (HPA: listed High cells; general IHC practice).
Unexpected cells stain more strongly than the intended reference cells.	Cell misidentification, nonspecific binding or endogenous detection activity are possibilities (general IHC practice); HPA's High list is not a complete negative list (HPA: tissue IHC).	Recheck morphology and the assay-matched negative control. Describe the observed cells and intensity without declaring the unlisted population AARS2-negative (general IHC practice; HPA: low tissue specificity).
Dense chromogen hides the intracellular pattern.	Overdevelopment can prevent useful compartment scoring (general IHC practice).	Review detection and development settings against the validated IHC procedure, then score a section where cytoplasm and nuclei remain distinguishable (general IHC practice).
What should an IF/ICC image show?	HPA reports enhanced mitochondrial ICC-IF localisation for both listed antibodies (HPA: subcellular ICC-IF and antibody validation).	Look for mitochondrial signal in the cell image; use the separate IF/ICC guide for its workflow. The ICC evidence supports localisation but supplies no paraffin IHC protocol (HPA: antibody validation).

Source: UniProt Q5JTZ9 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AARS2 staining

IHC analysis of AARS2 using anti-AARS2 antibody (A10160-2). AARS2 was detected in a paraffin-embedded section of human colon cancer 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 1:200 rabbit anti-AARS2 Antibody (A10160-2) 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.

A10160-2 · IHCIHC analysis of AARS2 using anti-AARS2 antibody (A10160-2). AARS2 was detected in a paraffin-embedded section of human colon cancer tissue.

IHC analysis of AARS2 using anti-AARS2 antibody (A10160-2). AARS2 was detected in a paraffin-embedded section of human colon 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 1:200 rabbit anti-AARS2 Antibody (A10160-2) 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.

A10160-2 · IHCIHC analysis of AARS2 using anti-AARS2 antibody (A10160-2). AARS2 was detected in a paraffin-embedded section of human colon tissue. Heat me

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124608-AARS2/tissue/cerebral+cortex#imid_8814995

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124608-AARS2/tissue/colon#imid_8816937

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124608-AARS2/tissue/liver#imid_8816954

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000124608-AARS2/tissue/kidney#imid_8816947

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035636; green = AARS2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for AARS2 IHC & IF

🧪Run breast first and score its glandular cells for AARS2 staining (HPA: High in breast glandular cells). HPA detects AARS2 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect adjacent stroma to lack specific glandular-pattern staining without treating its cells as proven AARS2-negative (HPA: no negative tissue listed; standard IHC practice).

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

Negative control tissue: None in HPA: AARS2 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 AARS2 in A-431, U-251MG, U2OS, HEK293, MCF-7, KOLF2.1J, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide and a rabbit IgG control matched to the primary antibody’s clonality, plus a matched AARS2-knockout sample if available (caption: rabbit primary; standard IHC specificity controls). Quench endogenous peroxidase before HRP/DAB detection and assess nonspecific background in the breast section (caption: HRP/DAB detection; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A10160-2 paraffin-section caption does not state a fixative (caption: fixative unreported). That caption reports heat retrieval in EDTA at pH 8.0 and primary antibody at 1:200 overnight at 4°C; it does not establish whether retrieval is required under other conditions (caption: IHC conditions). Relative ease of frozen-section IHC or IF is unreported; for breast sections, inspect glandular lumens and adjacent stroma for nonspecific DAB deposits when interpreting staining (standard IHC practice).

HPA tissue IHC evidence for AARS2

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: AARS2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced AARS2 IHC Tips

Use the catalog antibody’s paraffin-section IHC evidence to guide retrieval and detection, then assess AARS2 staining against its expected mitochondrial localisation.

Which retrieval conditions should I start with for AARS2 staining in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for AARS2 paraffin-section IHC (datasheet A10160-2). The selected colon cancer image used this retrieval with antibody at 1:200 overnight at 4°C (caption A10160-2). Keep heating conditions consistent across comparison sections, then check both DAB signal and tissue morphology. If staining is weak, compare a gentler EDTA cycle or citrate retrieval on adjacent sections while keeping antibody and detection conditions fixed. Judge any improvement by staining in intact cytoplasm, where tissue IHC shows AARS2 expression (HPA tissue IHC: general cytoplasmic expression).

How should I troubleshoot fixation-related loss of AARS2 staining?

The selected AARS2 paraffin-section caption does not report its fixative, so target-specific fixation sensitivity is unknown (caption A10160-2). Record the fixative, fixation duration and processing history for each specimen before comparing staining intensity. On serial sections from the same block, first apply the documented EDTA pH 8.0 retrieval and 1:200 antibody conditions (caption A10160-2). If similarly processed control sections stain but one specimen does not, inspect preservation and morphology before changing retrieval. Do not infer a particular fixation effect from the reported cytoplasmic tissue pattern (HPA tissue IHC: general cytoplasmic expression).

What AARS2 staining pattern should I expect in chromogenic tissue sections?

Interpret AARS2 DAB signal primarily within the cytoplasm: UniProt places the protein in mitochondria, and tissue IHC reports general cytoplasmic expression (UniProt Q5JTZ9: subcellular location; HPA tissue IHC: profile). Fine mitochondrial detail may be hard to resolve with chromogenic brightfield microscopy, so assess cellular distribution and morphology together. AARS2 has no annotated transmembrane segment; that annotation alone does not establish which mitochondrial compartment contains the epitope (UniProt Q5JTZ9: topology). Compare intact cells in the same section before calling a diffuse haze positive. Predominantly nuclear or extracellular signal warrants a specificity and background check against the expected mitochondrial location (UniProt Q5JTZ9: subcellular location).

Could AARS2 processing or an unknown epitope explain variable IHC staining?

The supplied record lists no annotated AARS2 isoforms and identifies a processed chain spanning residues 24–985 (UniProt Q5JTZ9: isoforms and processing). It also lists no annotated glycosylation sites or modified residues, and no transmembrane segment (UniProt Q5JTZ9: glycosylation, modified residues and topology). Those annotations do not identify the catalog antibody’s epitope or establish its accessibility after tissue processing. If staining varies, compare adjacent sections under the same EDTA pH 8.0 retrieval and 1:200 primary-antibody conditions (caption A10160-2). Treat any proposed epitope-masking explanation as provisional until it is supported by antibody-specific validation.

How can IF help assess an ambiguous AARS2 IHC pattern?

Use IF on a matched section to ask whether AARS2 signal overlaps a mitochondrial marker, consistent with its reported location (UniProt Q5JTZ9: subcellular location; HPA subcellular: mitochondria). In colon, multiplex with an endothelial marker such as CD31 when evaluating the cell population reported as highly stained by tissue IHC (HPA tissue IHC: colon endothelial cells, High). Choose a far-red fluorophore and include an unstained control to assess tissue autofluorescence. Test permeabilisation sufficient to reach the antibody’s epitope across mitochondrial membranes; the supplied topology does not specify its membrane-facing side (UniProt Q5JTZ9: topology). Compare localisation with the chromogenic result without assuming identical epitope access.

How do I distinguish AARS2 signal from nonspecific DAB background?

The selected paraffin-section example used 10% goat serum, 1:200 primary antibody overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (caption A10160-2). Start with those documented antibody and blocking conditions when evaluating excess staining. Include a no-primary control, and use a peroxidase block and controlled DAB development as general chromogenic IHC steps. Inspect pigment, section folds, edges and damaged tissue before assigning diffuse brown deposits to AARS2. Credible staining should track intact cellular cytoplasm and the expected mitochondrial distribution (HPA tissue IHC: profile; UniProt Q5JTZ9: subcellular location).

How should I score AARS2 IHC across tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION

Define cell populations and viable regions before scoring; tissue IHC reports high AARS2 staining in colon endothelial cells and low staining in adipocytes (HPA tissue IHC: colon and adipose tissue). For intensity, calculate an H-score by summing the percentages of cells at intensities 0–3 multiplied by their intensity, yielding 0–300. Report percent positive cells alongside that score, using the same positivity threshold throughout. If counting positive cells per mm², normalise to viable tissue area and state which cell population was counted. Keep retrieval, DAB development and image acquisition consistent across sections before interpreting score differences.

Which findings would make an apparent AARS2-positive section unconvincing?

Expect signal associated with cellular cytoplasm and mitochondria, because UniProt assigns AARS2 to mitochondria and tissue IHC describes general cytoplasmic staining (UniProt Q5JTZ9: subcellular location; HPA tissue IHC: profile). Strong signal confined to nuclei, extracellular debris or necrotic areas deserves review before it is scored. Check whether the stained cell population fits the section: colon endothelial cells are reported High, whereas adipocytes are reported Low (HPA tissue IHC: colon and adipose tissue). Edge-only staining and deposits persisting in a no-primary control suggest processing or detection artefact. Recheck peroxidase blocking and DAB development when brown signal appears independently of intact cell boundaries.

Boster reagents

Best AARS2 / Alanine--tRNA ligase, mitochondrial IHC Antibodies

A10160-2 has paraffin-section IHC images from human colon cancer and colon, and rat and mouse ovary (catalog IHC captions). No IF/ICC data are supplied (catalog applications; IF images).

Real IHC data

Anti-AARS2 Antibody

Cat # A10160-2

View product →

A10160-2 is listed for IHC in human, mouse and rat (catalog applications; reactivity). Its IHC captions show paraffin sections of human colon cancer, human colon, rat ovary and mouse ovary (catalog IHC captions).

Which to pick: Choose A10160-2 for paraffin-section tissue IHC: it is a rabbit polyclonal antibody with an IHC application and images from those tissues (catalog host; dilution record; applications; IHC captions). The captions do not report a fixative (catalog IHC captions). No listed SKU has an IF/ICC application or image, so there is no evidence-backed IF/ICC choice; for cross-species IHC, A10160-2 has human, mouse and rat reactivity and corresponding tissue images (catalog applications; IF images; reactivity; IHC captions).

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

References

UniProt Consortium. UniProt entry Q5JTZ9 (SYAM_HUMAN, Alanine--tRNA ligase, mitochondrial).
Human Protein Atlas. AARS2 tissue IHC expression (reliability: Approved).
Human Protein Atlas. AARS2 subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. AARS2 antibody validation summary (2 antibodies).
Integrative pan-cancer analysis reveals AARS2 as a lactylation-associated biomarker and therapeutic target in colon adenocarcinoma. Frontiers in immunology 2026 — PMC12982081.
AARS2 as a novel biomarker for prognosis and its molecular characterization in pan-cancer. Cancer medicine 2023 — PMC10726843.
Association of Hypoxia-Induced Lactate Accumulation with AARS2 Expression, PDHA1/CPT2 Lactylation, and Energy Metabolism in Yak Skeletal Muscle Cells. Cells 2026 — PMC13564726.
PubMed PMID:10574462 — UniProt-cited evidence.
PubMed PMID:14574404 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.