DARS2 / Aspartate--tRNA ligase, mitochondrial · IHC design guide

Design Immunohistochemistry for DARS2

Plan chromogenic DARS2 IHC on paraffin sections with the catalog antibody at 0.5–1 μg/mL (datasheet A06034-1). Interpret granular cytoplasmic staining against the reported tissue profile, bearing in mind its medium consistency with RNA expression (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for DARS2 (IHC for DARS2): expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial matrix/membrane (UniProt), antibody A06034-1, validated IHC image, and IHC protocol steps — Printable **DARS2** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial matrix/membrane (UniProt), antibody A06034-1, controls and protocol steps. Open the full DARS2 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

DARS2 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	Granular cytoplasm (HPA tissue IHC); mitochondrial matrix/membrane (UniProt)
	Staining pattern	Granular cytoplasmic staining in most tissues (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A06034-1)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	Heart muscle+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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06034-1)
	Caveat	Antibody staining and RNA show medium consistency (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	No isoforms annotated; residues 1–47 lie outside the mature chain (UniProt)

Section 1

Recommended DARS2 IHC & IF Protocols

The catalog antibody protocol (datasheet A06034-1) is followed by two published DARS2 IHC protocols using lung adenocarcinoma tissue (PMC10733159; PMC11535832).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet A06034-1)
Fixation	Image fixative and duration unreported (datasheet A06034-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A06034-1)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A06034-1)
Primary antibody	Rabbit anti-DARS2, 0.5-1μg/ml (datasheet A06034-1)
Primary incubation	Overnight at 4 °C (datasheet A06034-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A06034-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	DARS2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with heat retrieval in citrate buffer at pH 6, as specified for the catalog antibody and used in one published protocol (datasheet A06034-1; PMC10733159).

Section 2

What Is the Expected DARS2 Staining Pattern?

DARS2 localizes to the mitochondrial matrix and membrane and has no transmembrane segment (UniProt Q6PI48). In tissue sections, expect granular cytoplasmic staining, especially in glandular cells, kidney tubule cells and lung macrophages (HPA tissue IHC). HPA rates the tissue profile Enhanced, with medium consistency between staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic staining in colon or duodenum glandular cells, kidney tubules, or lung macrophages.	This fits the reported DARS2 pattern: each listed cell population stains at a High level, and the overall tissue profile is granular cytoplasmic (HPA tissue IHC). Compare staining within the named cells rather than treating every cell in the section as an equivalent positive control.
Predominantly nuclear, surface, or broad extracellular chromogen replaces the granular cytoplasmic pattern.	Treat this as a compartment mismatch that needs checking against controls: DARS2 is mitochondrial and lacks a transmembrane segment (UniProt Q6PI48). Additional nucleoplasmic localization is reported in ICC-IF, so isolated nuclear signal alone does not establish the expected tissue IHC pattern (HPA subcellular).
Strong staining appears mainly in cells scored as Not detected, such as cardiomyocytes or skeletal myocytes.	That differs from the named HPA observations and warrants checking cell identification, antibody specificity and chromogenic detection background (HPA tissue IHC; general IHC practice). An HPA 'Not detected' call describes its observed assay result; it does not prove biological absence under every condition.
Haze covers tissue and blank areas, obscuring cell boundaries and cytoplasmic granules.	Diffuse background cannot be scored as the reported granular cytoplasmic pattern (HPA tissue IHC). Review background controls, blocking, antibody concentration and wash conditions before interpreting apparent positives (general IHC practice). The pattern alone does not identify which step caused the haze.
No staining is visible in a section containing an expected High cell population.	A negative result in colon glandular cells, kidney tubules or lung macrophages conflicts with the listed HPA observations (HPA tissue IHC). Check that the relevant cells are present and that the staining run worked before calling DARS2 absent (general IHC practice).

💡Expected DARS2 appearanceCall a positive when identifiable High cells show granular cytoplasmic chromogen, especially glandular cells, kidney tubules or lung macrophages (HPA tissue IHC); diffuse, extracellular or nuclear-only color is a pattern mismatch requiring control review (UniProt Q6PI48; HPA subcellular; general IHC practice).

How each factor affects the staining
Cell population used for comparison	HPA reports High staining in several glandular populations, kidney tubule cells and lung macrophages, but Not detected in named muscle cells and fibroblasts (HPA tissue IHC). Score the specified cell type within its tissue; a tissue name alone is too broad a comparator.
Antibody validation	Both listed antibodies, HPA026506 and HPA026528, have Enhanced IHC validation (HPA antibodies). That supports the reported tissue pattern but does not make every unexpected signal specific. The overall tissue profile has medium staining-to-RNA consistency (HPA tissue IHC).
Protein location and processing	DARS2 is assigned to the mitochondrial matrix and membrane, with no transmembrane segment; its annotated chain spans residues 48–645 (UniProt Q6PI48). These annotations guide compartment interpretation but do not identify this antibody's epitope or establish an antigen-retrieval requirement.
Retrieval and chromogenic detection	Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.
What should IF/ICC show?	Mainly mitochondrial signal is supported; additional nucleoplasmic localization is approved in the ICC-IF record (HPA subcellular). This is an interpretation cue for the separate IF/ICC guide, not an IF/ICC protocol or a reason to score nuclear-only chromogen as the expected tissue IHC result.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells are blank.	The section may lack the intended cells, or the staining run may have failed (general IHC practice). HPA reports High staining in colon glandular cells, kidney tubules and lung macrophages (HPA tissue IHC).	Confirm the cell population on the counterstain, then examine a known-positive section processed in the same run and check the primary-antibody and detection steps (general IHC practice).
Color is strongest in nuclei.	The dominant tissue pattern disagrees with granular cytoplasmic IHC and mitochondrial localization (HPA tissue IHC; UniProt Q6PI48). HPA separately reports additional nucleoplasmic localization by ICC-IF (HPA subcellular).	Review a positive tissue control and background control, and record nuclear signal separately from granular cytoplasmic signal before assigning a DARS2-positive IHC score (general IHC practice).
Lung macrophages show broad, unusually intense chromogen.	Macrophages are a reported High population (HPA tissue IHC), while endogenous detection activity can also contribute chromogenic background in tissue (general IHC practice). Appearance alone cannot distinguish the two.	Inspect a detection-only control and the run's blocking controls; score DARS2 only when the staining pattern remains interpretable against those controls (general IHC practice).
Muscle cells or fibroblasts stain strongly.	Cardiomyocytes, skeletal myocytes, smooth muscle cells and soft-tissue fibroblasts are listed as Not detected in their respective HPA tissue observations (HPA tissue IHC). Cell misidentification or nonspecific signal is possible (general IHC practice).	Verify cell identity on the section and compare the signal with positive and detection-only controls before assigning it to DARS2 (general IHC practice).
Diffuse color makes granules hard to resolve.	The observed background does not match HPA's granular cytoplasmic profile; its source cannot be established from the slide alone (HPA tissue IHC; general IHC practice).	Check background controls, blocking, antibody concentration, washes and chromogen development, then reassess whether distinct cytoplasmic granules remain (general IHC practice).
Two sections give different apparent positivity.	A change in sampled cell populations can change the result because HPA staining levels vary by cell type (HPA tissue IHC). A run difference is also possible (general IHC practice).	Compare like cell populations first, then review the sections' controls and staining records. Report the cell type and compartment alongside intensity (general IHC practice).

Source: UniProt Q6PI48 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing DARS2 staining

IHC analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in paraffin-embedded section of human intestinal cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-DARS2 Antibody (A06034-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A06034-1 · IHCIHC analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in paraffin-embedded section of human intestinal cancer tissu

IHC analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-DARS2 Antibody (A06034-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A06034-1 · IHCIHC analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in paraffin-embedded section of human mammary cancer tissue.

IF analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-DARS2 Antibody (A06034-1) overnight at 4°C. DyLight®550 Conjugated Goat Anti-Rabbit IgG (BA1135) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A06034-1 · IFIF analysis of DARS2 using anti-DARS2 antibody (A06034-1). DARS2 was detected in immunocytochemical section of U20S cells. Enzyme antigen re

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117593-DARS2/tissue/cerebral+cortex#imid_6461288

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117593-DARS2/tissue/colon#imid_6461222

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117593-DARS2/tissue/liver#imid_6461223

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117593-DARS2/tissue/kidney#imid_6461216

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026528; green = DARS2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for DARS2 IHC & IF

🧪Run colon first: glandular cells should stain strongly (HPA: High in colon glandular cells). Use liver cholangiocytes as the negative tissue comparator (HPA: Not detected in liver cholangiocytes); on the colon slide, compare glandular staining with nonglandular cells that show only background signal, while treating their DARS2 status as unverified by the supplied HPA rows.

Positive control tissue: Appendix (Glandular cells, HPA High)

Negative control tissue: Heart muscle (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DARS2 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only section, a matched rabbit IgG isotype control (caption: rabbit anti-DARS2), and DARS2-knockout material if available as a biological negative (standard IHC controls). Block endogenous peroxidase and assess endogenous biotin before using the biotin-based DAB detection described for the positive tissue (caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected tissue-IHC caption does not state the fixative (caption: fixative not stated). The paraffin-section example used heat retrieval in citrate buffer at pH 6 for 20 minutes; this is a starting condition, while retrieval dependency across preparations is unreported (caption: heat-mediated antigen retrieval). There is no supplied comparison showing that frozen sections or IF are easier; in colon, assess mucus-associated background when interpreting chromogenic staining (HPA: colon glandular cells High; standard IHC practice).

HPA tissue IHC evidence for DARS2

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
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Epididymis	Glandular cells	High	Protein (IHC)	HPA →
Fallopian tube	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →
Skeletal muscle	Myocytes	Not detected	Protein (IHC)	HPA →
Smooth muscle	Smooth muscle cells	Not detected	Protein (IHC)	HPA →
Soft tissue	Fibroblasts	Not detected	Protein (IHC)	HPA →

Section 3

Advanced DARS2 IHC Tips

Troubleshoot DARS2 chromogenic IHC in paraffin sections using the catalog antibody’s tissue staining example and compartment specific controls.

How should I adjust retrieval when DARS2 staining is weak or diffuse?

Start with heat mediated citrate retrieval at pH 6 (datasheet A06034-1). The selected antibody stained a paraffin section after 20 minutes of retrieval in that buffer (caption A06034-1). If staining is weak, first check heating consistency and section adhesion, then compare a modestly longer retrieval time on matched sections while keeping detection conditions fixed (standard IHC practice). Score whether granular cytoplasmic staining improves without a parallel rise in diffuse background; that pattern is the expected tissue reference (HPA tissue IHC). Avoid treating stronger overall DAB signal alone as improved DARS2 detection (standard IHC practice).

Can fixation explain loss of DARS2 signal in paraffin sections?

Target specific fixation sensitivity is unknown: the selected paraffin section caption does not state its fixative (caption A06034-1). Record the actual fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining (standard IHC practice). Compare similarly processed sections using the same citrate pH 6 retrieval and detection settings to isolate fixation related variation (datasheet A06034-1; standard IHC practice). Include a section with established granular cytoplasmic staining, such as colon glandular cells, as a run control (HPA tissue IHC). Neither the reported tissue pattern nor DARS2 topology establishes how a particular fixative affects this antibody (HPA tissue IHC; UniProt Q6PI48).

What staining pattern should support DARS2 localisation in tissue?

Expect granular cytoplasmic staining in many tissues, consistent with the reported tissue IHC profile (HPA tissue IHC). DARS2 is assigned to the mitochondrial matrix and membrane, with no annotated transmembrane segment (UniProt Q6PI48). Assess granularity within intact cells at high magnification, and compare it with adjacent cells processed on the same slide (standard IHC practice). Strong glandular cell staining in colon or duodenum provides a tissue reference, while cardiomyocytes are reported as not detected (HPA tissue IHC). Predominantly smooth, diffuse cytoplasmic or uniformly nuclear DAB warrants review of background, morphology, and controls before assignment to DARS2 (UniProt Q6PI48; standard IHC practice).

Could epitope position or protein processing alter DARS2 IHC staining?

The record lists no isoforms and identifies residues 48–645 as the mature DARS2 chain (UniProt Q6PI48). Its mitochondrial location and lack of a transmembrane segment do not reveal which residues the catalog antibody recognizes (UniProt Q6PI48; caption A06034-1). Ask for the immunogen or mapped epitope before interpreting weak staining in altered specimens as loss of the full protein (standard IHC practice). Reported modifications include phosphothreonine 219, phosphoserine 242, and acetyllysines 368 and 382; their effect on this antibody is unestablished (UniProt Q6PI48). Compare staining with an independently validated antibody to a distinct epitope when epitope masking remains plausible (standard IHC practice).

How can IF help resolve an ambiguous chromogenic DARS2 pattern?

Use IF as a separate localisation check alongside the chromogenic IHC result; mitochondrial localisation is supported by the subcellular record (HPA subcellular). Multiplex DARS2 with a mitochondrial marker and, where cell identity is uncertain, a marker for the expected cell type, such as glandular cells in colon (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue autofluorescence before assigning puncta to DARS2 (standard IF practice). Because DARS2 is assigned to the mitochondrial matrix and membrane, assess permeabilisation that gives antibody access to the epitope’s side of mitochondrial membranes; the antibody epitope is unspecified (UniProt Q6PI48; standard IF practice). Keep IF controls and interpretation distinct from the paraffin IHC example (caption A06034-1; standard IF practice).

How do I reduce background without erasing granular DARS2 staining?

The selected paraffin example used 10% goat serum blocking, 1 μg/mL primary antibody overnight at 4°C, and biotin based DAB detection (caption A06034-1). Check a no primary control and block endogenous peroxidase before DAB development to identify detection background (standard IHC practice). With biotin based detection, assess endogenous biotin if staining persists in the no primary control (caption A06034-1; standard IHC practice). Titrate primary concentration and development time on matched sections while retaining citrate pH 6 retrieval (datasheet A06034-1; standard IHC practice). Preserve the granular cytoplasmic pattern reported across tissues rather than optimizing for the darkest slide (HPA tissue IHC).

How should I score DARS2 staining across samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue region before scoring, because DARS2 staining varies by cell type (HPA tissue IHC; standard IHC practice). For intact glandular cells, record the percentage at each staining intensity and calculate an H-score; also report the percentage positive cells when intensity grading is unreliable (standard IHC practice). Normalize positive counts to the number of eligible cells, or positive cell density to viable tissue area in mm² (standard IHC practice). Exclude necrosis, folds, and section edges using the same rules across samples (standard IHC practice). Keep retrieval, DAB development, imaging, and scoring thresholds consistent, and report granular cytoplasmic signal separately from nonspecific diffuse staining (HPA tissue IHC; standard IHC practice).

When is a DARS2 positive call credible rather than artefactual?

A credible call shows granular cytoplasmic signal in intact cells, matching the reported tissue pattern and mitochondrial localisation (HPA tissue IHC; UniProt Q6PI48). High staining in colon glandular cells or lung macrophages is compatible with the tissue reference, whereas cardiomyocytes are reported as not detected (HPA tissue IHC). Review predominantly nuclear signal cautiously: an additional nucleoplasmic location is reported in cellular imaging, but the tissue IHC reference is granular cytoplasmic (HPA subcellular; HPA tissue IHC). Edge restricted staining, necrotic debris, and signal reproduced without primary antibody favor artefact (standard IHC practice). Use a no primary control and assess endogenous peroxidase or biotin contributions when interpreting this biotin based DAB workflow (caption A06034-1; standard IHC practice).

Boster reagents

Best DARS2 / Aspartate--tRNA ligase, mitochondrial IHC Antibodies

A06034-1 has human paraffin-section IHC images and IF/ICC images in U20S and A431 cells (catalog image captions); its listed reactivity is human (catalog: reactivity).

Real IHC data

Anti-DARS2 Antibody ®

Cat # A06034-1

View product →

A06034-1 is listed for human IHC and IF/ICC (catalog: applications and reactivity). Its IHC captions show human intestinal, mammary and lung cancer paraffin sections; its IF captions show U20S and A431 cells (catalog image captions).

Which to pick: Choose A06034-1 for tissue IHC: its own images document paraffin sections, and its listed IHC concentration is 0.5–1 μg/ml (catalog: IHC captions and dilution). For IF/ICC, the same SKU lists those applications and 2 μg/ml, with images of U20S and A431 cells (catalog: applications, IF dilution and captions). No cross-species option is listed because reactivity is human only; clonality and the IHC fixative are unreported (catalog: reactivity, clone field and IHC captions).

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

References

UniProt Consortium. UniProt entry Q6PI48 (SYDM_HUMAN, Aspartate--tRNA ligase, mitochondrial).
Human Protein Atlas. DARS2 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. DARS2 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nucleoplasm..
Human Protein Atlas. DARS2 antibody validation summary (2 antibodies).
Prognostic biomarker DARS2 correlated with immune infiltrates in bladder tumor. Frontiers in immunology 2023 — PMC10827901.
DARS2 promotes the occurrence of lung adenocarcinoma via the ERK/c-Myc signaling pathway. Thoracic cancer 2023 — PMC10733159.
Upregulation of DARS2 by HBV promotes hepatocarcinogenesis through the miR-30e-5p/MAPK/NFAT5 pathway. Journal of experimental & clinical cancer research : CR 2017 — PMC5649064.
Role of CENPL, DARS2, and PAICS in determining the prognosis of patients with lung adenocarcinoma. Translational lung cancer research 2024 — PMC11535832.
PubMed PMID:16710414 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:15779907 — UniProt-cited evidence.