SLC25A6 / ADP/ATP translocase 3 · IHC design guide

Design Immunohistochemistry for SLC25A6

Plan paraffin-section SLC25A6 IHC using the granular cytoplasmic tissue pattern (HPA tissue IHC). Compare cardiomyocytes with high staining against epididymal glandular cells with no detected staining (HPA tissue IHC), while accounting for possible detection of products from more than one gene (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for SLC25A6 (IHC for SLC25A6): expected localisation Granular cytoplasm (HPA tissue IHC); inner mitochondrial membrane (UniProt), antibody A09457-1, validated IHC image, and IHC protocol steps — Printable **SLC25A6** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); inner mitochondrial membrane (UniProt), antibody A09457-1, controls and protocol steps. Open the full SLC25A6 IHC guide →

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SLC25A6 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); inner mitochondrial membrane (UniProt)
	Staining pattern	Granular cytoplasmic staining in most tissues (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen)
	Positive control ⓘ	Cerebellum+4 more · see all
	Negative control ⓘ	Epididymis+3 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 A09457-1)
	Caveat	Antibody may detect products of more than one gene (HPA tissue IHC)
	Regulation	No specific expression regulator reported (UniProt)
	Isoform / epitope	No isoforms; N-terminal processing may affect an N-terminal epitope (UniProt)

Section 1

Recommended SLC25A6 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with one published SLC25A6 tissue-section protocol (datasheet; PMC13468794 methods).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A09457-1)
Fixation	Image fixative and duration unreported (datasheet A09457-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-SLC25A6, 1:50-1:200 (datasheet A09457-1)
Primary incubation	Overnight at 4 °C (standard)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SLC25A6-positive staining in purkinje cells of cerebellum (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min for this membrane antigen (page retrieval rule; UniProt P12236 localization). The published protocol does not specify retrieval (PMC13468794 methods).

Section 2

What Is the Expected SLC25A6 Staining Pattern?

SLC25A6 is an inner mitochondrial membrane protein with six transmembrane segments (UniProt P12236 topology). In paraffin-section IHC, expect granular cytoplasmic staining in many tissues, with high staining reported in cardiomyocytes, Purkinje cells, and several glandular cell populations (HPA tissue IHC). Interpret that pattern cautiously: HPA rates its tissue IHC evidence Approved, with medium consistency between staining and RNA data, and warns that the antibodies target proteins from more than one gene (HPA tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic stain in cardiomyocytes or Purkinje cells, with little nuclear stain (HPA tissue IHC).	This fits the reported high staining in those cells and a mitochondrial location (HPA tissue IHC; UniProt P12236). Morphology alone cannot establish that every granule contains SLC25A6, given HPA's multi-gene antibody warning (HPA tissue IHC).
Predominantly nuclear or uniform membrane-edge stain in otherwise intact cells.	That distribution conflicts with the expected granular cytoplasmic pattern and inner mitochondrial membrane location (HPA tissue IHC; UniProt P12236). Review morphology and controls before assigning it to the target; the location evidence does not establish this pattern as SLC25A6 (HPA tissue IHC).
Strong stain in epididymal glandular cells or ovarian stromal cells.	HPA reports these specific cell populations as not detected (HPA tissue IHC). Consider cross-reactivity, endogenous chromogen-producing activity, or a specimen-specific result (HPA multi-gene antibody warning; general IHC practice). An HPA negative is a reference observation, not proof that every specimen must be negative (HPA tissue IHC).
Broad, diffuse color across cells and extracellular areas obscures cytoplasmic granules.	The slide cannot be scored confidently against HPA's granular cytoplasmic profile (HPA tissue IHC). Diffuse background can arise from nonspecific binding or detection chemistry; assess the reagent controls and staining distribution before changing the biological interpretation (general IHC practice).
No convincing stain in heart-muscle cardiomyocytes or cerebellar Purkinje cells.	Both are reported as high-staining reference cells (HPA tissue IHC). First suspect a staining-run or specimen issue and compare controls; absence in one section alone does not overturn the tissue profile, especially given its medium staining–RNA consistency (HPA tissue IHC; general IHC practice).

💡Expected SLC25A6 appearanceCall a slide pattern-consistent when the expected cells show granular cytoplasmic staining, especially high staining in cardiomyocytes, Purkinje cells, or listed glandular cells; dominant nuclear stain or diffuse color is suspect (HPA tissue IHC; UniProt P12236).

How each factor affects the staining
Cell and tissue selection	High staining is reported in heart-muscle cardiomyocytes, cerebellar Purkinje cells, rectal and stomach glandular cells, and testicular Leydig cells (HPA tissue IHC). HPA reports lower or undetected staining in other named cell populations, so score the cell type as well as the tissue (HPA tissue IHC).
Subcellular expectation	The protein has six transmembrane segments in the mitochondrial inner membrane (UniProt P12236 topology). HPA describes tissue staining as granular cytoplasmic and ICC-IF localization as mitochondrial (HPA tissue IHC; HPA subcellular). Neither source defines an antibody epitope or an antigen-retrieval setting.
Antibody evidence and specificity	HPA rates the tissue IHC profile Approved, with medium staining–RNA consistency, and cautions that antibodies target proteins from more than one gene (HPA tissue IHC). Its listed IHC antibody, HPA071684, is Approved; the listed ICC antibody, HPA046835, is Supported for ICC (HPA antibodies).
Processing and variants	UniProt lists full-length residues 1–298 and an N-terminally processed form, residues 2–298, with no annotated isoforms or glycosylation sites (UniProt P12236). These annotations do not predict a different tissue staining pattern or a retrieval response without epitope-specific evidence.
IF/ICC Q: Should fluorescence appear in mitochondria?	Yes. HPA reports approved mitochondrial localization in ICC-IF images from MCF-7, PC-3, and U2OS cells, while warning that the antibodies target proteins from multiple genes (HPA subcellular). This supports a localization expectation; it supplies no IF/ICC protocol for this IHC section.
Chromogenic background	Endogenous enzyme activity and nonspecific reagent binding can add color during chromogenic IHC (general IHC practice). Use appropriate detection controls to distinguish that color from the reported cell-specific granular cytoplasmic pattern (general IHC practice; HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A high-staining reference tissue shows no signal.	The run may have failed, or the selected section may not show the relevant cells (general IHC practice; HPA tissue IHC).	Check that cardiomyocytes or Purkinje cells are present, then review the positive control, primary-antibody step, detection reagents, and counterstain before judging the tissue negative (HPA tissue IHC; general IHC practice).
Signal is diffuse or masks cell boundaries.	Nonspecific binding or detection background can obscure HPA's granular cytoplasmic profile (general IHC practice; HPA tissue IHC).	Inspect a no-primary control and the distribution of color; review blocking, washes, primary-antibody concentration, and detection conditions as general IHC troubleshooting steps (general IHC practice).
Nuclear stain dominates an otherwise positive section.	A nuclear-dominant pattern does not match the reported mitochondrial or granular cytoplasmic localization (UniProt P12236; HPA tissue IHC).	Compare the suspect cells with an expected positive cell population and the no-primary control; avoid calling the nuclear signal SLC25A6 solely from stain intensity (HPA tissue IHC; general IHC practice).
A reported undetected cell population stains strongly.	Cross-reactivity or detection background is plausible, particularly because HPA cautions that antibodies target proteins from more than one gene (HPA tissue IHC; general IHC practice).	Verify the cell identity, compare a high-staining reference on the same run, and inspect detection controls; record the discrepancy rather than treating HPA's undetected call as an absolute biological rule (HPA tissue IHC; general IHC practice).
Only an IF/ICC image is available for comparison.	HPA's mitochondrial ICC-IF localization and its tissue IHC profile come from different assay contexts, with separate antibody validation entries (HPA subcellular; HPA antibodies).	Use IF/ICC to assess the expected compartment, then judge the paraffin-section result against tissue cell types and granular cytoplasmic staining reported for IHC (HPA subcellular; HPA tissue IHC).
Changing antigen retrieval changes the apparent pattern.	The supplied UniProt topology and HPA staining profile do not establish SLC25A6-specific retrieval sensitivity (UniProt P12236; HPA tissue IHC).	Compare conditions using the same positive tissue and appropriate controls, and report the observed result as an assay finding rather than a documented target-specific retrieval effect (HPA tissue IHC; general IHC practice).

Source: UniProt P12236 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SLC25A6 staining

A09457-1 · IHCImmunohistochemistry (IHC) analyzes of SLC25A6 (R155) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169100-SLC25A6/tissue/cerebral+cortex#imid_19730395

HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000169100-SLC25A6/tissue/small+intestine#imid_19730296

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169100-SLC25A6/tissue/colon#imid_19730329

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169100-SLC25A6/tissue/liver#imid_19730322

HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA046835; green = SLC25A6, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for SLC25A6 IHC & IF

🧪Run heart muscle first: cardiomyocytes should stain (HPA: High in cardiomyocytes). Use ovary as the negative tissue, assessing ovarian stroma cells (HPA: Not detected in ovarian stroma cells); on the heart slide, acellular extracellular matrix should remain free of specific chromogen (standard IHC practice).

Positive control tissue: Cerebellum (Purkinje cells, HPA High)

Negative control tissue: Epididymis (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SLC25A6 in MCF-7, PC-3, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use a matched SLC25A6 knockout specimen as a biological negative if available (standard IHC practice). Quench endogenous peroxidase and check pigment in heart sections so intrinsic color is not scored as staining (standard IHC practice).

⚠️Feasibility: The supplied evidence reports no target-specific fixation window or retrieval dependence; the A09457-1 paraffin-section caption gives 1:50 for human breast carcinoma but does not report a fixative (selected IHC caption: 1:50; fixative unreported). Optimize antigen retrieval empirically for IHC-P; the evidence does not establish whether frozen sections or IF/ICC are easier (supplied evidence: no matched comparison). For IF/ICC, assess a mitochondrial pattern while accounting for the HPA caution that its localization summary draws on antibodies targeting proteins from multiple genes (HPA: mitochondria, approved; HPA subcellular caution).

HPA tissue IHC evidence for SLC25A6

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Cerebellum	Purkinje cells	High	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	High	Protein (IHC)	HPA →
Parathyroid gland	Glandular cells	High	Protein (IHC)	HPA →
Rectum	Glandular cells	High	Protein (IHC)	HPA →
Stomach	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Epididymis	Glandular cells	Not detected	Protein (IHC)	HPA →
Ovary	Ovarian stroma cells	Not detected	Protein (IHC)	HPA →
Soft tissue	Chondrocytes	Not detected	Protein (IHC)	HPA →
Vagina	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced SLC25A6 IHC Tips

Use compartment, cell type and matched controls to troubleshoot SLC25A6 staining in chromogenic IHC; the IF/ICC considerations appear in one entry.

How should I retrieve SLC25A6 in paraffin sections when staining is weak?

Begin with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule). Cool sections consistently, then compare retrieved and untreated serial sections while holding antibody dilution and chromogen development constant (standard IHC practice). The selected product image documents paraffin-section staining at 1:50, but its caption gives no retrieval conditions, so it cannot establish which treatment produced that image (A09457-1 caption). If signal remains weak, test a different retrieval buffer as a fallback on matched sections; assess tissue damage and background alongside granular cytoplasmic staining (standard IHC practice; HPA tissue IHC profile).

Could fixation explain weak or uneven SLC25A6 staining?

Target-specific fixation sensitivity is unknown: the selected tissue caption identifies paraffin sections but does not state the fixative or fixation time (A09457-1 caption). Record fixative, duration, section age and retrieval history for each case, then compare matched sections processed together before changing antibody concentration (standard IHC practice). Use the page’s citrate pH 6.0 retrieval at 95–98 °C for 20 min as the common starting condition (page retrieval rule). If staining varies across a block, inspect morphology and section edges, and repeat on a better-preserved area where available; neither a tissue staining pattern nor the protein’s membrane topology establishes a fixation effect (standard IHC practice).

What staining pattern supports SLC25A6 localisation in tissue sections?

Expect fine granular cytoplasmic staining in appropriate cells, consistent with the tissue IHC profile and the protein’s mitochondrial inner-membrane location (HPA tissue IHC profile; UniProt P12236 subcellular location). Chromogenic resolution generally cannot resolve the inner mitochondrial membrane, so assess the distribution of cytoplasmic granules rather than claiming membrane-level localisation from DAB alone (standard IHC interpretation). Compare adjacent cells and a matched control section, including an omission control, while checking whether diffuse nuclear or luminal colour follows the same pattern (standard IHC practice). Interpret agreement cautiously because the HPA tissue antibody can recognise proteins from more than one gene (HPA tissue IHC reliability description).

How can epitope position affect interpretation of SLC25A6 staining?

Map the antibody’s documented immunogen against the SLC25A6 sequence before attributing a change in staining to epitope loss (standard IHC practice). The record lists a 298-residue chain, an N-terminally processed 2–298 chain and 6 transmembrane segments; it lists 0 alternative isoforms (UniProt P12236 processing, topology and isoforms). Reported modifications include N-terminal acetylation and modified lysines at positions 52, 105 and 268, but their effect on this antibody is unspecified (UniProt P12236 modified residues; A09457-1 caption). If an epitope is undocumented, compare a second well-characterised antibody and suitable controls before assigning different tissue patterns to processing or modification (standard IHC practice).

How should I check an IF pattern against the chromogenic IHC result?

Use IF/ICC as a separate localisation check, pairing the SLC25A6 signal with a marker for the expected cell type and a mitochondrial marker where appropriate (standard IF practice; UniProt P12236 subcellular location). Choose a fluorophore channel after measuring tissue autofluorescence in an unstained section, and include single-colour controls before interpreting overlap (standard IF practice). Match permeabilisation to the documented epitope: matrix-facing or intermembrane-space-facing regions require access across different mitochondrial membranes, and the epitope of the selected antibody is not specified here (UniProt P12236 topology; A09457-1 caption). HPA reports mitochondrial IF localisation, but warns that its antibodies target proteins from multiple genes; that result does not validate this catalog antibody for IF (HPA subcellular summary).

What should I check when DAB background obscures SLC25A6 granules?

Run an antibody-omission section and inspect whether brown deposit persists, especially near blood, damaged tissue or section edges (standard IHC practice). Apply a peroxidase block before HRP/DAB detection, optimise protein blocking and washes, and shorten chromogen development if the control is also coloured (standard chromogenic IHC practice). If background appears only with primary antibody, compare serial sections with a lower concentration than the 1:50 used in the selected paraffin-section image, while keeping retrieval and detection constant (A09457-1 caption; standard IHC practice). Judge improvement by preservation of discrete cytoplasmic granules rather than by overall darkness, and retain the same counterstain across comparisons (HPA tissue IHC profile; standard IHC practice).

How should I score SLC25A6 staining across comparable sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and scoring area before reviewing treatment groups, then score granular cytoplasmic staining separately from diffuse background (HPA tissue IHC profile; standard IHC practice). For cell-based analysis, record the percentage of positive cells and intensity categories to calculate an H-score; for spatial analysis, report positive-cell density per mm² (standard IHC quantification practice). Normalise counts to viable tissue area or the number of eligible cells in the same compartment, excluding folds and necrotic regions by a preset rule (standard IHC quantification practice). Keep exposure, DAB development, thresholds and counterstain consistent, and report the antibody and retrieval conditions with the scores (standard IHC practice).

When is a positive SLC25A6 IHC result convincing?

A convincing result shows reproducible granular cytoplasmic staining in intact cells, compatible with SLC25A6’s mitochondrial inner-membrane location and the reported tissue pattern (UniProt P12236 subcellular location; HPA tissue IHC profile). Compare expected positive populations, such as cardiomyocytes with reported high staining, against a matched control rather than assigning identity from brown colour alone (HPA tissue IHC: high in cardiomyocytes; standard IHC practice). Treat isolated nuclear or luminal staining, section-edge accentuation and staining confined to necrosis as suspect until controls support them (standard IHC interpretation). Persistent colour in an antibody-omission section suggests endogenous enzyme or detection background, while HPA’s multi-gene antibody caution limits gene-specific conclusions from staining alone (standard IHC practice; HPA tissue IHC reliability description).

Boster reagents

Best SLC25A6 / ADP/ATP translocase 3 IHC Antibodies

A09457-1 has a real IHC image from paraffin-embedded human breast carcinoma (image caption). The catalog lists human, mouse and rat reactivity; IF/ICC is listed for A09457-2 without figure data (catalog).

Real IHC data Immunohistochemistry (IHC) analyzes of SLC25A6 (R155) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.

Anti-SLC25A6 (R155) Antibody

Cat # A09457-1

View product →

The rendered A09457-1 card shows IHC in paraffin-embedded human breast carcinoma at 1:50 (image caption). A09457-2 is listed for IHC and IF/ICC in human and mouse, but has no IHC or IF figure in the payload (catalog: applications, reactivity, image alts).

Which to pick: Choose A09457-1 for tissue IHC because its own image documents a paraffin section; the caption does not report the fixative (image caption). Choose A09457-2 for IF/ICC based on its listed applications, while treating imaging conditions as unverified here because no IF figure is supplied (catalog: applications, image alts). Both are rabbit polyclonals; for rat samples, choose A09457-1 because only it lists rat reactivity (catalog: host, dilution_raw, reactivity).

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

References

UniProt Consortium. UniProt entry P12236 (ADT3_HUMAN, ADP/ATP translocase 3).
Human Protein Atlas. SLC25A6 tissue IHC expression (reliability: Approved).
Human Protein Atlas. SLC25A6 subcellular location (ICC-IF): Localized to the mitochondria. Caution: Based on antibodies targeting proteins from multiple genes..
Human Protein Atlas. SLC25A6 antibody validation summary (2 antibodies).
DNMT3B- SLC25A6 axis-mediated DNA methylation regulates ferroptosis to promote paclitaxel resistance in triple-negative breast cancer. Frontiers in cell and developmental biology 2026 — PMC13468794.
MRPL13 enhances mitochondrial function and promotes tumor progression in ovarian cancer by inhibiting mPTP opening via SLC25A6. Cell death & disease 2025 — PMC12371087.
Comprehensive Analysis and Validation of Solute Carrier Family 25 (SLC25) and Its Correlation with Immune Infiltration in Pan-Cancer. BioMed research international 2022 — PMC9569204.
PubMed PMID:2541251 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:2829183 — UniProt-cited evidence.

Immunohistochemistry protocol sheet for SLC25A6 (ADP/ATP translocase 3). Expected localisation Granular cytoplasm (HPA tissue IHC); inner mitochondrial membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A09457-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Purkinje cells); negative Epididymis. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A09457-1); 2. Fixation: Image fixative and duration unreported (datasheet A09457-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-SLC25A6, 1:50-1:200 (datasheet A09457-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: 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 A09457-1); Caveat: Antibody may detect products of more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms; N-terminal processing may affect an N-terminal epitope (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt P12236, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria. Caution: Based on antibodies targeting proteins from…, Datasheet A09457-1, PMC13468794. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SLC25A6/immunohistochemistry-ihc-guide.