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ACADM / Medium-chain specific acyl-CoA dehydrogenase, mitochondrial · IHC design guide
Design Immunohistochemistry for ACADM
Plan chromogenic IHC on paraffin sections using the catalog antibody’s IHC conditions. Compare granular cytoplasmic staining (HPA tissue IHC) with ACADM’s mitochondrial matrix location (UniProt), and include positive and negative cell populations when interpreting signal.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACADM 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 in tissue (HPA tissue IHC); mitochondrial matrix (UniProt) | |
| Staining pattern | Granular cytoplasmic staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02383-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02383-2) | |
| Caveat | Adipocytes may lack detectable staining (HPA tissue IHC) | |
| Regulation | Highest expression in heart and muscle (UniProt) | |
| Isoform / epitope | 2 isoforms; mature chain spans residues 26–421; epitope map unknown (UniProt) |
Section 1
Recommended ACADM IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: A02383-2). One published ACADM IHC protocol provides a citrate retrieval alternative (PMC13297011).
| Sample | Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet A02383-2) |
| Fixation | Image fixative and duration unreported (datasheet A02383-2); 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 A02383-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02383-2) |
| Primary antibody | Rabbit anti-ACADM, 2 μg/ml (datasheet A02383-2) |
| Primary incubation | Overnight at 4 °C (datasheet A02383-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02383-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACADM-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: A02383-2); try citrate pH 6.0 when adapting the published protocol (PMC13297011).
Section 2
What Is the Expected ACADM Staining Pattern?
ACADM is a mitochondrial matrix protein with no transmembrane segment (UniProt P11310: location and topology). In paraffin-section IHC, expect granular cytoplasmic staining across many cell types, with high staining in heart cardiomyocytes and several glandular or epithelial populations (HPA: Enhanced tissue IHC reliability; high in listed cells). HPA describes medium consistency between antibody staining and RNA expression, so judge a result by its cellular and subcellular pattern as well as intensity (HPA: reliability description).
| Granular cytoplasmic staining in heart cardiomyocytes or adrenal glandular cells. | This fits the reported high staining in both cell populations and the ubiquitous granular cytoplasmic tissue pattern (HPA: tissue IHC). The granules are consistent with mitochondrial localization, although chromogenic IHC alone does not identify individual organelles (UniProt P11310: matrix; general IHC interpretation). |
| Predominantly nuclear or cell-surface staining, with little granular cytoplasmic signal. | This conflicts with the reported tissue pattern and matrix localization (HPA: tissue IHC; UniProt P11310: location and topology). Treat it as suspect staining and assess controls before assigning it to ACADM (general IHC practice). |
| Strong staining concentrated in adipocytes, basal-layer skin cells, or soft-tissue fibroblasts. | HPA reports ACADM as not detected in those specific cell populations (HPA: tissue IHC). Unexpected strong staining warrants a cross-reactivity or endogenous-detection check; a single positive cell does not establish either cause (general IHC practice). |
| A uniform haze across cells and surrounding section, obscuring cytoplasmic granules. | The expected HPA pattern is granular and cytoplasmic, so diffuse section-wide color cannot be scored confidently as ACADM (HPA: tissue IHC). Review blocking, washes, detection controls, and counterstain intensity (general IHC practice). |
| No staining in heart cardiomyocytes while the section and detection controls are evaluable. | This disagrees with HPA's high cardiomyocyte staining (HPA: heart muscle IHC). Check antibody identity and dilution, retrieval conditions, and the detection sequence before interpreting another tissue as ACADM-negative (general IHC practice). |
💡Expected ACADM appearanceCall a positive result when high, granular cytoplasmic staining appears in heart cardiomyocytes or HPA-listed glandular cells; dominant nuclear, cell-surface, or uniform section-wide color is suspect (HPA: tissue IHC; UniProt P11310: mitochondrial matrix; general IHC interpretation).
| Subcellular location and topology (UniProt P11310). | ACADM resides in the mitochondrial matrix and has no transmembrane segment (UniProt P11310). Interpret tissue IHC as cytoplasmic granularity; do not require a membrane outline (HPA: granular cytoplasmic profile; general IHC interpretation). |
| Tissue and cell-type baseline (HPA: tissue IHC; UniProt P11310: tissue specificity). | HPA reports high cardiomyocyte, adrenal glandular, and respiratory epithelial staining, but no detected signal in listed adipocytes, basal-layer skin cells, or fibroblasts (HPA: tissue IHC). UniProt reports ubiquitous expression with highest levels in heart and muscle (UniProt P11310). |
| Antibody validation and expression agreement (HPA: antibody and tissue IHC records). | HPA006198 and HPA026542 each have Enhanced IHC validation, while the tissue profile has medium staining-to-RNA consistency (HPA: antibody validation; tissue reliability). Interpret a discordant field with its controls and cell identity in view (general IHC practice). |
| Processing and isoforms (UniProt P11310). | UniProt annotates a mature chain at residues 26–421 and 2 isoforms (UniProt P11310). The payload gives no antibody epitope, so it cannot establish whether an isoform or the removed N-terminal region changes staining (source limitation). |
| IF/ICC Q: What localization would corroborate the IHC pattern? | A: Predominantly mitochondrial fluorescence is supported by HPA ICC-IF and agrees with UniProt's matrix annotation (HPA: supported main mitochondrial location; UniProt P11310). HPA also reports additional perinuclear-theca, calyx, and principal-piece localization in its subcellular record (HPA: approved additional locations). |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive heart control has little or no signal (HPA: high cardiomyocyte staining). | The run may have inadequate antibody or detection performance; the pattern alone cannot identify which step failed (general IHC practice). | Check the catalog antibody's IHC-P instructions, antibody dilution, retrieval and detection steps, then repeat with the heart control (general IHC practice). |
| The section shows mostly nuclear or membrane-edge color (HPA: granular cytoplasmic profile). | This differs from mitochondrial matrix localization; nonspecific primary binding or detection background are possibilities (UniProt P11310; general IHC practice). | Compare a known-positive tissue and primary-omission control, then reassess the staining compartment before scoring (general IHC practice). |
| Adipocytes stain as strongly as the positive cells (HPA: adipocytes not detected). | The cell-type mismatch may reflect cross-reactivity or endogenous detection activity; HPA's negative call alone does not determine which (HPA: tissue IHC; general IHC practice). | Confirm cell identity and compare primary-omission and detection controls; score the expected granular signal separately (general IHC practice). |
| Diffuse brown background hides the granules (HPA: granular cytoplasmic profile). | Incomplete blocking, insufficient washing, or excess detection signal can reduce contrast (general IHC practice). | Review blocking and wash steps and compare a primary-omission control; adjust detection conditions according to the assay controls (general IHC practice). |
| Signal varies between tissue types despite an acceptable heart control (HPA: high cardiomyocyte staining). | HPA reports different staining levels by cell type and only medium agreement with RNA data (HPA: tissue IHC reliability). | Compare matched cell populations with HPA's cell-specific calls; avoid treating an entire tissue as uniformly positive or negative (HPA: tissue IHC; general IHC interpretation). |
| A retrieval change alters signal in a repeat run (general IHC practice). | The supplied HPA and UniProt records do not establish ACADM-specific retrieval or fixation sensitivity (source limitation). | Compare conditions using the same known-positive tissue and controls; record the tested conditions without attributing the change to a documented ACADM fixation effect (general IHC practice). |
Experiment evidence
IHC & IF images showing ACADM staining
A02383-2 · IHCIHC analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-2). ACADM/MCAD was detected in a paraffin-embedded section of human bladde
A02383-2 · IHCIHC analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-2). ACADM/MCAD was detected in a paraffin-embedded section of human breast
A02383-2 · IFIF analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-2). ACADM/MCAD was detected in an immunocytochemical section of Hela cells.
A02383-3 · IHCIHC analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-3). ACADM/MCAD was detected in a paraffin-embedded section of human colore
A02383-3 · IHCIHC analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-3). ACADM/MCAD was detected in a paraffin-embedded section of human lung c
A02383-3 · IFIF analysis of ACADM/MCAD using anti-ACADM/MCAD antibody (A02383-3). ACADM/MCAD was detected in an immunocytochemical section of Hela cells.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117054-ACADM/tissue/cerebral+cortex#imid_1962292
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117054-ACADM/tissue/colon#imid_6615294
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117054-ACADM/tissue/liver#imid_6615295
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117054-ACADM/tissue/kidney#imid_1962212
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA026542; green = ACADM, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA026542; green = ACADM, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACADM IHC & IF
🧪Run colon first and score its glandular cells, where ACADM staining is High (HPA: colon, glandular cells, High). Use soft-tissue fibroblasts as the negative tissue (HPA: soft tissue, fibroblasts, Not detected); on the colon slide, assess morphologically distinct nonglandular cells as internal background references only where they are unstained, since their ACADM status is not specified by the supplied HPA row.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACADM in U-251MG, U2OS, A-431, Sperm, KOLF2.1J, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and rabbit isotype controls matched to the primary antibody’s clonality where known, plus ACADM-knockout material as a biological negative (selected-SKU caption: rabbit primary; clonality unreported). Block endogenous peroxidase and check endogenous biotin background in the colon section when using the caption’s biotin–streptavidin/DAB detection (selected-SKU caption: biotinylated secondary, streptavidin–biotin complex and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 was used in that IHC example, but whether ACADM staining depends on that retrieval condition is unreported (selected-SKU tissue-IHC caption: EDTA pH 8.0 retrieval). The supplied evidence does not establish that frozen sections or IF are easier; for colon chromogenic IHC, assess background from endogenous peroxidase and the biotin-based detection system with the controls above (selected-SKU caption: streptavidin–biotin/DAB detection).
HPA tissue IHC evidence for ACADM
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced ACADM IHC Tips
Troubleshoot ACADM staining in paraffin sections by checking retrieval, mitochondrial localisation, controls, and cell-specific scoring (UniProt P11310; HPA tissue IHC).
What retrieval should I try when ACADM staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections stained with A02383-2 (datasheet A02383-2). Keep section thickness, heating, cooling, and antibody incubation consistent while comparing a weak section with a known positive control (standard IHC practice). The selected image used 2 μg/ml primary antibody overnight at 4°C, followed by biotinylated secondary antibody and DAB detection (datasheet A02383-2). If staining remains weak, compare a second retrieval condition on adjacent sections while monitoring tissue damage and background; the supplied evidence establishes EDTA pH 8.0 as the starting condition (datasheet A02383-2).
Could fixation explain weak or uneven ACADM staining?
The selected paraffin-section caption does not state its fixative, so target-specific fixation sensitivity for ACADM is unknown (datasheet A02383-2). Record fixative, fixation duration, tissue thickness, and processing history for each specimen before attributing a staining difference to ACADM biology (standard IHC practice). Compare sections processed together, using the documented EDTA pH 8.0 retrieval and 2 μg/ml antibody condition as a reference (datasheet A02383-2). Patchy staining beside poorly preserved tissue can reflect processing variation; inspect morphology and repeat with matched controls before assigning biological meaning (standard IHC practice). Neither granular HPA staining nor mitochondrial localisation establishes how this antigen responds to a particular fixative (HPA tissue IHC; UniProt P11310).
Where should convincing ACADM staining appear in a paraffin section?
Expect a granular cytoplasmic pattern in positive cells (HPA tissue IHC), consistent with ACADM in the mitochondrial matrix (UniProt P11310). The protein has no annotated transmembrane segment, so a crisp plasma-membrane outline would conflict with its recorded topology (UniProt P11310 topology). Evaluate staining within intact cells alongside morphology and a matched negative control; diffuse extracellular deposits or staining confined to section edges warrant technical review (standard IHC practice). Cardiomyocytes and several listed glandular or epithelial cell populations have high HPA staining and can provide tissue context, whereas basal-layer skin cells are listed as not detected (HPA tissue IHC).
How do isoforms and epitope uncertainty affect ACADM IHC?
UniProt lists 2 ACADM isoforms, but the supplied antibody caption does not define the recognized epitope or establish isoform selectivity (UniProt P11310; datasheet A02383-2). Interpret positive staining as antibody reactivity in tissue, rather than a measurement of one isoform, unless independent epitope information becomes available (standard IHC interpretation). ACADM is processed to a chain spanning residues 26–421 and has several annotated modified residues, including acetylation and succinylation (UniProt P11310). Those annotations do not show whether this antibody recognizes a processing-dependent or modification-dependent epitope; compare matched sections and controls before interpreting a staining change as altered expression (UniProt P11310; standard IHC practice).
How should an ACADM IHC result guide a separate IF experiment?
Use the IHC result to select cells for an IF comparison, then multiplex ACADM with a marker of the expected cell type; HPA reports high staining in cardiomyocytes (HPA tissue IHC). Choose a fluorophore channel with low tissue autofluorescence and include unstained and single-label controls to assess bleed-through (standard IF practice). ACADM is a mitochondrial matrix protein without an annotated transmembrane segment, so access to a matrix-facing epitope requires suitable permeabilisation; the antibody epitope itself is unspecified (UniProt P11310 topology; datasheet A02383-2). Check for punctate mitochondrial overlap against the cell marker and controls, while optimising IF fixation and permeabilisation independently because the supplied IHC caption does not specify a fixative (HPA subcellular; datasheet A02383-2).
How can I reduce diffuse or granular background in ACADM IHC?
The selected IHC image used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and a biotin-based DAB detection sequence (datasheet A02383-2). Run a primary-omission control and inspect whether staining persists after secondary and detection reagents; block endogenous peroxidase as part of the chromogenic workflow (standard IHC practice). If background rises across many cell types, optimise antibody concentration, washing, and DAB development with matched positive tissue rather than judging intensity alone (standard IHC practice). ACADM can produce granular cytoplasmic staining across tissues (HPA tissue IHC), so preserve a plausible cellular pattern while reducing diffuse deposits and edge staining (standard IHC practice).
How should I score ACADM staining across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then report the percentage of positive cells and an intensity-based H-score for cytoplasmic staining (standard IHC practice). If spatial burden matters, count positive cells per mm² of viable tissue and normalise to the number of eligible cells or viable area, using the same denominator across samples (standard IHC practice). Record granular signal within intact cells separately from diffuse pigment, necrosis, and section-edge staining (HPA tissue IHC; standard IHC practice). Keep retrieval, antibody concentration, detection time, exposure to DAB, and scoring thresholds consistent; the selected caption provides EDTA pH 8.0 retrieval and 2 μg/ml antibody as documented conditions (datasheet A02383-2).
When is an apparent ACADM-positive cell likely to be an artefact?
A credible positive cell has granular cytoplasmic staining, compatible with the HPA tissue pattern and mitochondrial matrix localisation (HPA tissue IHC; UniProt P11310). Question signal confined to nuclei, plasma membranes, necrotic areas, or section edges, and compare those regions with morphology and a primary-omission control (UniProt P11310 topology; standard IHC practice). In DAB IHC, residual endogenous peroxidase or detection-related deposits can mimic brown signal; verify the peroxidase block and inspect the negative control (standard IHC practice). Interpret cell identity carefully: HPA lists high staining in cardiomyocytes but no detectable staining in adipocytes, while its tissue IHC reliability is Enhanced with medium agreement between staining and RNA (HPA tissue IHC).
Boster reagents
Best ACADM / Medium-chain specific acyl-CoA dehydrogenase, mitochondrial IHC Antibodies
A02383-2 and A02383-3 have human paraffin-section IHC images and HeLa-cell IF images; both list human, mouse, and rat reactivity (catalog image captions; catalog reactivity).
Real IHC data
Anti-ACADM/MCAD Antibody ®
Cat # A02383-2
Real IHC data
Anti-ACADM/MCAD Antibody ®
Cat # A02383-3
A02383-2 has IHC images from human bladder epithelial carcinoma, breast carcinoma, Classic Hodgkin’s lymphoma, and hepatocellular carcinoma; A02383-3 has images from human colorectal and lung cancers (catalog IHC captions). Both have HeLa-cell IF images, while M02383 and M02383-2 list IHC and IF applications and human, mouse, and rat reactivity without supplied images (catalog IF captions; catalog applications and reactivity).
Which to pick: For tissue IHC, choose A02383-2 or A02383-3 according to the illustrated human tissue: each caption documents paraffin sections, EDTA pH 8.0 retrieval, and DAB detection; the fixative is unreported (catalog IHC captions). For IF/ICC, either has a HeLa-cell image using 5 μg/mL primary antibody (catalog IF captions). For mouse or rat samples, all four list reactivity, but the supplied IHC and IF images show human samples only; M02383 and M02383-2 are listed as rabbit monoclonals without supplied images (catalog reactivity, clones, and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P11310 (ACADM_HUMAN, Medium-chain specific acyl-CoA dehydrogenase, mitochondrial).
- Human Protein Atlas. ACADM tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ACADM subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the perinuclear theca, calyx and principal piece..
- Human Protein Atlas. ACADM antibody validation summary (2 antibodies).
- Low ACADM expression predicts poor prognosis and suppressive tumor microenvironment in clear cell renal cell carcinoma. Scientific reports 2024 — PMC11045743.
- Low expression of fatty acid oxidation related gene ACADM indicates poor prognosis of renal clear cell carcinoma and is related to tumor immune infiltration. Oncology research 2024 — PMC10865730.
- Schisandrin B Attenuates Renal Fibrotic Remodeling in Association with Restoration of a PPARα-Related Tubular Fatty-Acid Oxidation Program. Biomedicines 2026 — PMC13297011.
- Three novel hub genes and their clinical significance in clear cell renal cell carcinoma. Journal of Cancer 2019 — PMC6909945.
- PubMed PMID:3035565 — UniProt-cited evidence.
- PubMed PMID:1731887 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACADM (Medium-chain specific acyl-CoA dehydrogenase, mitochondrial). Expected localisation Granular cytoplasm in tissue (HPA tissue IHC); mitochondrial matrix (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02383-2). Validated IHC antibody: A02383-2 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet A02383-2); 2. Fixation: Image fixative and duration unreported (datasheet A02383-2); 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 A02383-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02383-2); 8. Primary antibody: Rabbit anti-ACADM, 2 μg/ml (datasheet A02383-2); 9. Primary incubation: Overnight at 4 °C (datasheet A02383-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02383-2); Caveat: Adipocytes may lack detectable staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain spans residues 26–421 ; epitope map unknown (UniProt); Regulation: Highest expression in heart and muscle (UniProt). Evidence: UniProt P11310, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A02383-2, PMC13297011. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACADM/immunohistochemistry-ihc-guide.