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ACADS / Short-chain specific acyl-CoA dehydrogenase, mitochondrial · IHC design guide
Design Immunohistochemistry for ACADS
Plan chromogenic ACADS IHC around granular cytoplasmic staining in hepatocytes and glandular cells (HPA tissue IHC). For paraffin sections, the IHC-validated antibody has a stated range of 2–5 μg/mL (datasheet A05028-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACADS 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 with a mitochondrial pattern (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasm in hepatocytes and glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05028-1) | |
| Positive control | Colon+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 paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Endogenous liver peroxidase can mimic DAB staining (standard IHC practice) | |
| Regulation | RNA enhanced: liver, skeletal muscle (HPA tissue IHC: RNA specificity) | |
| Isoform / epitope | No annotated isoforms; mature chain spans residues 25–412 (UniProt) |
Section 1
Recommended ACADS IHC & IF Protocols
The catalog antibody’s IHC protocol (datasheet A05028-1) is followed by two published ACADS chromogenic IHC protocols (PMC13022253; PMC11351355).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05028-1) |
| Fixation | Image fixative and duration unreported (datasheet A05028-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A05028-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05028-1) |
| Primary antibody | Rabbit anti-ACADS, 2-5μg/ml (datasheet A05028-1) |
| Primary incubation | Overnight at 4 °C (datasheet A05028-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05028-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACADS-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular, mitochondrial pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A05028-1); citrate pH 6.0 was used in one published protocol (PMC13022253).
Section 2
What Is the Expected ACADS Staining Pattern?
ACADS is a soluble mitochondrial matrix enzyme with no transmembrane segment (UniProt P16219 topology). In paraffin-section IHC, expect granular cytoplasmic staining consistent with mitochondria (HPA: tissue IHC profile; reliability Enhanced, with medium consistency between staining and RNA). HPA reports high staining in hepatocytes, intestinal glandular cells, kidney collecting ducts, lung type II alveolar cells, and testicular Leydig cells (HPA: tissue IHC).
| Granular cytoplasmic staining in hepatocytes or intestinal glandular cells. | This fits the mitochondrial pattern and cells with high observed staining (HPA: tissue IHC). Judge the pattern within cells as well as its strength; HPA’s Enhanced reliability still carries a medium staining–RNA consistency caveat (HPA: reliability description). |
| Predominantly nuclear or diffuse whole-cell signal, without cytoplasmic granules. | This is discordant with the expected paraffin IHC pattern (HPA: tissue IHC; UniProt P16219: mitochondrial matrix). Review specificity and image interpretation before calling it ACADS. HPA lists uncertain additional nucleoplasmic localization in ICC-IF, so isolated nuclear signal is not by itself a validated IHC pattern (HPA: subcellular). |
| Strong staining in fibroblasts while expected positive cells stain weakly. | HPA lists fibroblasts in soft tissue as low staining, not as a proven negative control (HPA: tissue IHC). This reversal raises concern for cross-reactivity or endogenous chromogenic activity (general IHC practice); assess the control slide and tissue context before assigning either cause. |
| Uniform haze across cells and surrounding tissue obscures granules. | Diffuse background prevents a reliable mitochondrial call (HPA: expected granular tissue IHC profile). Possible general IHC causes include nonspecific antibody binding or incomplete control of endogenous detection activity; compare a matched no-primary control before scoring (general IHC practice). |
| No detectable signal in hepatocytes on a run expected to be positive. | Hepatocytes have high observed staining (HPA: liver tissue IHC), so a blank result calls for a technical check before a biological interpretation. Check the run’s positive control, detection reagents, and tissue integrity (general IHC practice); HPA does not establish a target-specific fixation cause. |
💡Expected ACADS appearanceCall a convincing positive when high-staining cells such as hepatocytes show granular cytoplasmic signal consistent with mitochondria (HPA: tissue IHC); dominant nuclear signal or broad diffuse haze is suspect (HPA: tissue IHC; general IHC interpretation).
| Cell choice and intensity | HPA reports high protein staining in colon, duodenum, rectum, and small-intestine glandular cells; kidney collecting ducts; hepatocytes; lung type II alveolar cells; and Leydig cells (HPA: tissue IHC). Its tissue-enhanced RNA call for liver and skeletal muscle is a separate measurement, not a claim of high IHC staining in every cell there (HPA: RNA specificity). |
| Compartment and processing | The mature annotated chain spans residues 25–412, and ACADS resides in the mitochondrial matrix without a transmembrane segment (UniProt P16219). Use a granular cytoplasmic distribution to assess localization (HPA: tissue IHC); these annotations do not identify an antibody epitope or prescribe retrieval conditions. |
| Antibody evidence | HPA022271 and CAB019284 have Enhanced IHC validation; HPA004799 has no stated IHC validation in this payload (HPA: antibody records). Enhanced supports the reported tissue pattern through independent antibodies or orthogonal data (HPA: antibody validation definition), while the overall staining–RNA consistency is medium (HPA: reliability description). |
| IF/ICC Q&A: What localization should a separate IF assay assess? | Mainly mitochondria (supported); nucleoplasm and centrosome are uncertain additional locations, while sperm mid, principal, and end pieces are approved additional locations (HPA: subcellular ICC-IF). Those ICC-IF observations inform localization interpretation; this paraffin IHC section supplies no IF protocol (HPA: subcellular). |
| Chromogenic detection controls | Endogenous enzyme activity can produce color independently of primary-antibody binding (general IHC practice). A no-primary control helps distinguish that activity or detection background from an ACADS pattern; it cannot alone prove antibody specificity (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue is blank, including hepatocytes. | A run-level detection problem or insufficient assay sensitivity is possible (general IHC practice); hepatocytes are high-staining in HPA tissue IHC. | Verify the positive-control slide and detection reagents, then review the antibody’s IHC-P instructions for its specified retrieval and dilution (general IHC practice). No ACADS-specific retrieval or dilution value is supplied here. |
| All compartments stain evenly in expected positive tissue. | The result lacks the granular mitochondrial pattern reported for ACADS (HPA: tissue IHC); nonspecific binding or detection background is possible (general IHC practice). | Compare a matched no-primary control, assess blocking and chromogen development, and score ACADS only where the expected cellular pattern remains discernible (general IHC practice; HPA: tissue IHC). |
| Nuclei dominate the stain while cytoplasmic granules are absent. | The pattern conflicts with the main mitochondrial assignment (UniProt P16219; HPA: subcellular). HPA’s additional nucleoplasmic ICC-IF location is uncertain (HPA: subcellular). | Check morphology, counterstain, and control slides; seek concordant granular staining with an IHC-validated antibody before interpreting the nuclear signal as ACADS (general IHC practice; HPA: antibody records). |
| Fibroblasts appear stronger than nearby expected positive cells. | Soft-tissue fibroblasts are listed as low in HPA tissue IHC. Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare cell identities and a no-primary control, then repeat assessment with an IHC-validated antibody if needed (general IHC practice; HPA: antibody records). Do not use fibroblasts as an absolute negative control. |
| Only a small subset of cells stains within a listed tissue. | HPA reports staining for specified cell populations, such as kidney collecting ducts and lung type II alveolar cells, rather than every cell in those organs (HPA: tissue IHC). | Identify the stained cell population on the counterstained section before judging the tissue as discordant (general IHC practice); compare it with the cell-level HPA entry, not just the organ name. |
| An ICC-IF image suggests a different compartment than the paraffin IHC slide. | HPA’s main ICC-IF location is mitochondria, with additional locations carrying different confidence labels (HPA: subcellular); the supplied IHC profile is granular cytoplasmic (HPA: tissue IHC). | Interpret each assay against its own reported pattern and validation (HPA: tissue IHC; HPA: subcellular). For this IHC slide, prioritize cell-level granular staining and investigate a dominant discordant pattern with controls (general IHC practice). |
Experiment evidence
IHC & IF images showing ACADS staining
A05028-1 · IHCIHC analysis of ACADS/SCAD using anti-ACADS/SCAD antibody (A05028-1). ACADS/SCAD was detected in a paraffin-embedded section of human colon
A05028-1 · IHCIHC analysis of ACADS/SCAD using anti-ACADS/SCAD antibody (A05028-1). ACADS/SCAD was detected in a paraffin-embedded section of human liver
A05028-1 · IFIF analysis of ACADS/SCAD using anti-ACADS/SCAD antibody (A05028-1). ACADS/SCAD was detected in immunocytochemical section of A549 cells. En
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000122971-ACADS/tissue/cerebral+cortex#imid_5782652
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000122971-ACADS/tissue/colon#imid_5782586
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000122971-ACADS/tissue/liver#imid_5782587
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000122971-ACADS/tissue/kidney#imid_5782580
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA022271; green = ACADS, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA022271; green = ACADS, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACADS IHC & IF
🧪Start with colon: glandular cells should stain strongly (HPA: High in colon glandular cells). HPA lists no negative tissue and detects ACADS in all 45 scored tissues (HPA); use no-primary and isotype controls for the negative comparison, and treat any weakly stained non-glandular cells on the positive slide only as internal contrast, not as proven ACADS-negative cells.
Positive control tissue: Colon (Glandular cells, HPA High)
Negative control tissue: None in HPA: ACADS 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 ACADS in Hep-G2, RT-4, SK-MEL-30, JURKAT, Sperm, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only section and a concentration-matched nonimmune rabbit IgG isotype control alongside the test section (selected-SKU caption: rabbit primary antibody); use ACADS knockout material or an immunizing-peptide block, if available, to test specificity. Quench endogenous peroxidase and compare background in colon sections before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section caption reports EDTA retrieval at pH 8.0 and a primary concentration of 2 μg/ml overnight at 4°C; it does not report a fixative or establish whether retrieval is required under other conditions (selected-SKU tissue-IHC caption). A target-specific fixation window and fixation effect are unreported in the supplied evidence, and the evidence does not establish that frozen sections or IF are easier. Expect a mitochondrial staining pattern (UniProt P16219: mitochondrial matrix; HPA: mitochondrial localization supported), while checking colon sections for endogenous peroxidase background during chromogenic detection (standard IHC practice).
HPA tissue IHC evidence for ACADS
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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ACADS is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ACADS IHC Tips
Troubleshoot ACADS staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring against the documented IHC result and tissue expression profile.
Which retrieval condition should I start with for ACADS in paraffin sections?
Start with heat-mediated retrieval in EDTA, pH 8.0, for the catalog antibody in paraffin sections (datasheet A05028-1). Keep the retrieval vessel, heating time and cooling time consistent across sections, and record them before comparing staining intensity (standard IHC practice). The documented human colon cancer section was blocked with 10% goat serum, then incubated with 2 μg/ml primary antibody overnight at 4°C (datasheet A05028-1). If staining remains weak, test an alternative retrieval condition on adjacent sections while retaining the EDTA-treated section as the reference and checking for preserved morphology (standard IHC practice).
Can the available evidence identify the best fixative for ACADS staining?
The catalog image documents a paraffin-embedded human colon cancer section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A05028-1). Do not attribute weak staining to a particular fixative from the tissue image alone; compare sections with documented fixation histories under the same EDTA, pH 8.0 retrieval condition (datasheet A05028-1; standard IHC practice). Include a positive control with a reproducible cellular pattern in each run, and inspect morphology before changing antibody concentration (standard IHC practice). Record fixation duration, processing and section thickness when comparing runs because these variables can change apparent staining and tissue preservation (standard IHC practice).
What cellular pattern should count as plausible ACADS staining?
Look for granular cytoplasmic staining consistent with mitochondria, rather than treating diffuse staining alone as confirmation of ACADS (HPA tissue IHC: granular mitochondrial pattern; UniProt P16219: mitochondrial matrix). The protein has no annotated transmembrane segment, so a crisp cell-surface rim is discordant with its recorded topology (UniProt P16219). Compare glandular cells in colon or duodenum, hepatocytes in liver, or kidney collecting ducts with surrounding cells to judge whether the pattern is biologically plausible (HPA tissue IHC: High in these cells). Inspect adjacent sections at the same magnification and detection exposure, since chromogen intensity by itself cannot establish subcellular localisation (standard IHC practice).
How should epitope uncertainty affect interpretation of a weak ACADS stain?
No alternative isoforms or mapped antibody epitope are supplied here, so weak staining cannot be assigned to isoform selectivity or a particular masked residue (UniProt P16219: 0 annotated isoforms; datasheet A05028-1: epitope unspecified). ACADS is recorded as a 412-residue precursor with a mature chain spanning residues 25–412 (UniProt P16219). Several modified residues are annotated, including threonine 27 and lysines 51 and 72, but their effect on this antibody is unreported (UniProt P16219; datasheet A05028-1). Troubleshoot retrieval and detection with matched control sections before proposing an epitope-based explanation (standard IHC practice).
How can IF help assess the mitochondrial pattern seen by chromogenic IHC?
Use the separate IF/ICC workflow to compare ACADS with a mitochondrial marker and, when needed, a marker identifying the expected cell population (UniProt P16219: mitochondrial matrix; HPA tissue IHC: cell-specific expression; standard IF practice). Choose fluorophores and filter sets after checking tissue autofluorescence, with unstained and single-color controls to distinguish background and spectral overlap (standard IF practice). Because the expected epitope lies within the mitochondrial matrix, optimize permeabilisation for antibody access while preserving organelle structure; the antibody epitope itself is not mapped in the supplied evidence (UniProt P16219; datasheet A05028-1; standard IF practice). HPA supports mitochondrial localisation in its subcellular data, but that observation does not specify a fixation or permeabilisation recipe for this antibody (HPA subcellular).
How do I distinguish weak ACADS signal from DAB background?
Run a section without primary antibody alongside the test section to assess secondary and chromogen background (standard IHC practice). The documented workflow used 10% goat serum blocking, a peroxidase-conjugated secondary and DAB detection after the primary incubation (datasheet A05028-1). Add an endogenous peroxidase block and control DAB development time as general chromogenic IHC steps, particularly when background obscures granular cytoplasmic detail (standard IHC practice). Compare staining in the expected cellular compartments with diffuse haze, pigment and staining at tissue edges before changing the 2 μg/ml documented primary concentration (HPA tissue IHC: granular mitochondrial pattern; datasheet A05028-1; standard IHC practice).
What should an ACADS IHC score measure across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: for example, score colon glandular cells separately from neighboring cells and record granular cytoplasmic signal (HPA tissue IHC: High in colon glandular cells; HPA tissue IHC: granular mitochondrial pattern). An H-score combines the percentage of cells in each intensity category with their intensity, while percentage-positive scoring reports the fraction above a prespecified threshold (standard IHC practice). Normalize counts or positive area to viable tissue area in mm², or to the number of eligible cells, and exclude necrosis and section folds by a consistent rule (standard IHC practice). Use the same retrieval, DAB development and imaging settings across compared sections so technical variation does not masquerade as expression change (standard IHC practice).
When should an apparent ACADS-positive cell be treated as suspect?
A convincing result shows granular cytoplasmic staining in an appropriate cell population, consistent with mitochondrial matrix localisation and the tissue profile (UniProt P16219; HPA tissue IHC). Strong cell-surface or predominantly nuclear staining needs independent review: the protein has no annotated transmembrane segment, while the HPA nucleoplasmic location is marked uncertain (UniProt P16219; HPA subcellular). Treat staining restricted to section edges, necrotic areas or cells outside the prespecified population cautiously, and compare it with the no-primary control (standard IHC practice). Residual endogenous peroxidase can also generate DAB signal, so confirm that the peroxidase block and negative control behaved as expected before calling a sample positive (standard IHC practice).
Boster reagents
Best ACADS / Short-chain specific acyl-CoA dehydrogenase, mitochondrial IHC Antibodies
A05028-1 has paraffin-section IHC images from human colon and liver cancer tissue and an IF image from A549 cells (catalog image captions). Human, mouse and rat are listed as reactive species (catalog).
Real IHC data
Anti-ACADS/SCAD Antibody ®
Cat # A05028-1
A05028-1 is the only SKU that will render; its IHC images show human colon and liver cancer paraffin sections (catalog IHC image captions). Its IF image shows A549 cells (catalog IF image caption).
Which to pick: Choose A05028-1 for tissue IHC when an image-supported paraffin-section example matters: its caption describes EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A05028-1 IHC image caption). Choose A05028-1 for IF/ICC because both applications are listed and an A549 IF image is provided; M05028-1 lists IHC but has no IHC image or IF/ICC application (catalog). Both SKUs list human, mouse and rat reactivity, while only M05028-1 is identified as monoclonal; the supplied tissue images establish human paraffin-section examples only (catalog; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P16219 (ACADS_HUMAN, Short-chain specific acyl-CoA dehydrogenase, mitochondrial).
- Human Protein Atlas. ACADS tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ACADS subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nucleoplasm, centrosome, mid piece, principal piece and end piece..
- Human Protein Atlas. ACADS antibody validation summary (3 antibodies).
- Short-chain acyl-CoA dehydrogenase initiates mtDNA demethylation and leakage to fuel antitumor immunity in colorectal cancer. Signal transduction and targeted therapy 2026 — PMC13022253.
- Sodium butyrate mediates histone crotonylation and alleviated neonatal rats hypoxic-ischemic brain injury through gut-brain axis. Frontiers in microbiology 2022 — PMC9631217.
- Acetylome Analyses Provide New Insights into the Effect of Chronic Intermittent Hypoxia on Hypothalamus-Dependent Endocrine Metabolism Impairment. Biology 2024 — PMC11351355.
- DNA methylation of ACADS promotes immunogenic cell death in hepatocellular carcinoma. Cell & bioscience 2025 — PMC11727568.
- PubMed PMID:2565344 — UniProt-cited evidence.
- PubMed PMID:9383286 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACADS (Short-chain specific acyl-CoA dehydrogenase, mitochondrial). Expected localisation Granular cytoplasm with a mitochondrial pattern (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05028-1). Validated IHC antibody: A05028-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05028-1); 2. Fixation: Image fixative and duration unreported (datasheet A05028-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A05028-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05028-1); 8. Primary antibody: Rabbit anti-ACADS, 2-5μg/ml (datasheet A05028-1); 9. Primary incubation: Overnight at 4 °C (datasheet A05028-1). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Endogenous liver peroxidase can mimic DAB staining (standard IHC practice); Isoform / epitope: No annotated isoforms; mature chain spans residues 25–412 (UniProt); Regulation: RNA enhanced: liver, skeletal muscle (HPA tissue IHC: RNA specificity). Evidence: UniProt P16219, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A05028-1, PMC13022253, PMC11351355. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACADS/immunohistochemistry-ihc-guide.