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TOMM40 / Mitochondrial import receptor subunit TOM40 homolog · IHC design guide
Design Immunohistochemistry for TOMM40
Plan TOMM40 paraffin IHC around the granular cytoplasmic tissue pattern (HPA tissue IHC). Use strongly stained glandular or epithelial cells as positive references and interpret the pattern alongside TOMM40’s outer mitochondrial membrane location (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TOMM40 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 outer membrane (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in glandular and epithelial cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03166-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Soft tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Low macrophage staining may be missed (HPA tissue IHC) | |
| Regulation | Specific regulation is not annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended TOMM40 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A03166-2). The published tissue microarray protocol below uses citrate retrieval (PMC13380028).
| Sample | Paraffin-embedded human diffuse large B cell lymphoma tissue; fixative not specified (datasheet A03166-2) |
| Fixation | Image fixative and duration unreported (datasheet A03166-2); 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 A03166-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03166-2) |
| Primary antibody | Rabbit anti-TOMM40, 2-5 μg/ml (datasheet A03166-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03166-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03166-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TOMM40-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet A03166-2); compare citrate pH 6.0 during optimization (PMC13380028).
Section 2
What Is the Expected TOMM40 Staining Pattern?
TOMM40 resides in the mitochondrial outer membrane, where UniProt annotates multiple membrane-spanning segments (UniProt O96008 topology). In paraffin-section IHC, expect granular cytoplasmic staining, especially in glandular cells of the colon, appendix, rectum and small intestine, among other HPA high-staining cells (HPA: tissue IHC). HPA rates the tissue staining pattern Supported, with high consistency between antibody staining and RNA expression (HPA: reliability Supported).
| Granular cytoplasmic staining in colon or small-intestine glandular cells (HPA: High in glandular cells). | This fits the reported tissue pattern and mitochondrial outer-membrane location (HPA: general granular cytoplasmic pattern; UniProt O96008: outer membrane). Judge signal within cells against a counterstain; a brown deposit alone does not establish its compartment (standard IHC practice). |
| Predominantly nuclear, cell-surface or extracellular staining, with little granular cytoplasmic signal. | Those compartments do not match the expected IHC pattern (HPA: granular cytoplasmic pattern; UniProt O96008: outer membrane). Consider nonspecific binding, detection artefact or a localization error; the slide alone cannot identify which. Review morphology and appropriate controls before assigning TOMM40 expression (standard IHC practice). |
| Strong staining in chondrocytes while expected positive glandular cells remain faint or unstained (HPA: chondrocytes Not detected; glandular cells High). | That reversal warrants a specificity check, including possible antibody cross-reactivity or endogenous detection activity (standard IHC practice). HPA's chondrocyte result is an observed reference pattern, not proof that every chondrocyte in every specimen must be negative (HPA: soft-tissue chondrocytes Not detected). |
| Diffuse chromogen across tissue, stroma and cell-free areas obscures cytoplasmic granules. | Uniform background is difficult to reconcile with the reported cell-associated granular pattern (HPA: tissue IHC profile). Review blocking, washes and detection controls as general IHC checks; this appearance cannot by itself establish whether TOMM40 is present (standard IHC practice). |
| No convincing signal in colon or appendix glandular cells (HPA: High in both). | A negative result in an HPA high-staining reference population merits a technical check before biological interpretation (HPA: tissue IHC). Confirm tissue morphology, control staining, antibody dilution and detection performance; HPA's observed intensity does not guarantee the same intensity in every preparation (standard IHC practice). |
💡Expected TOMM40 appearanceCall a positive result when glandular cells show clear granular cytoplasmic staining, potentially strong in HPA high-staining tissues; predominantly nuclear or diffuse cell-free deposits argue against the expected pattern (HPA: tissue IHC; UniProt O96008: outer membrane).
| Compartment and topology (UniProt O96008 topology; HPA: tissue IHC profile). | TOMM40 is an outer-mitochondrial-membrane protein, while tissue IHC reports a granular cytoplasmic appearance. Read granules within the cytoplasm rather than expecting a continuous outline of each mitochondrion in a chromogenic section (UniProt O96008: outer membrane; HPA: granular cytoplasmic pattern). |
| Reference-cell selection (HPA: tissue IHC). | HPA reports High staining in several glandular populations and skin basal-layer cells, but Low staining in lung macrophages, smooth-muscle cells, skeletal myocytes and adipocytes. These levels guide comparison between cell types; they do not set a universal intensity cutoff for individual slides (HPA: tissue IHC). |
| Antibody evidence (HPA: antibody validation). | HPA lists IHC as Supported for HPA036231 and HPA036232, both rabbit polyclonal antibodies (HPA: antibody records). That supports comparison with the reported tissue pattern; it does not establish that an unlisted catalog antibody has identical specificity or staining intensity (HPA: antibody records). |
| Isoforms and processing (UniProt O96008). | UniProt records two isoforms and a chain spanning residues 1–361, with no signal peptide, propeptide or annotated glycosylation sites (UniProt O96008: isoforms and processing). The supplied evidence does not identify the IHC antibody epitope or establish isoform-specific staining, so avoid assigning a granule to either isoform. |
| IF/ICC Q&A: What localization is expected? (HPA: subcellular ICC-IF). | Mainly mitochondrial signal, with an additional cytosolic location reported by HPA (HPA: mitochondria enhanced; cytosol approved). This provides a localization cross-check for IF/ICC; it does not specify an IF/ICC protocol or change the granular cytoplasmic interpretation of paraffin-section IHC (HPA: subcellular ICC-IF; tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known positive glandular cells show no stain (HPA: colon and appendix glandular cells High). | The stain or detection run may have failed; absence on one slide cannot establish TOMM40 loss (standard IHC practice). | Check a positive control and the detection run, then verify antibody dilution against its supplied IHC instructions (standard IHC practice). |
| Signal is weak but confined to cytoplasmic granules (HPA: granular cytoplasmic pattern). | Expression varies by cell population: HPA reports Low staining in several cell types (HPA: tissue IHC). | Compare the same cell type with an HPA high-staining reference and assess morphology before calling the run negative (HPA: tissue IHC; standard IHC practice). |
| Most nuclei are stained more strongly than cytoplasm. | A predominantly nuclear pattern conflicts with the expected mitochondrial and granular cytoplasmic localization; its precise cause remains undetermined (UniProt O96008: outer membrane; HPA: tissue IHC). | Check counterstain and detection controls, then reassess localization with the IHC-validated antibody if needed (standard IHC practice; HPA: IHC Supported). |
| Chondrocytes stain strongly (HPA: chondrocytes Not detected). | Possible nonspecific antibody binding or endogenous detection activity; HPA's reference result alone cannot distinguish them (HPA: tissue IHC; standard IHC practice). | Review a reagent-omission control and the staining of expected positive cells in the same run (standard IHC practice; HPA: glandular cells High). |
| Diffuse deposit obscures cell borders and granules. | Background from blocking, washing or detection can conceal the reported granular cytoplasmic pattern (standard IHC practice; HPA: tissue IHC profile). | Inspect a detection control and review blocking and wash steps before scoring cell-associated signal (standard IHC practice). |
| Two antibodies give different staining patterns (HPA: HPA036231 and HPA036232 IHC Supported). | Supported IHC status does not guarantee identical staining in every preparation (HPA: antibody records). | Compare their cell-type and compartment patterns against HPA references, using matched controls; treat discordant localization as unresolved (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing TOMM40 staining
A03166-2 · IHCIHC analysis of TOMM40 using anti-TOMM40 antibody (A03166-2). TOMM40 was detected in a paraffin-embedded section of human diffuse large B ce
A03166-2 · IHCIHC analysis of TOMM40 using anti-TOMM40 antibody (A03166-2). TOMM40 was detected in a paraffin-embedded section of human liver cancer tissu
A03166-2 · IFIF analysis of TOMM40 using anti-TOMM40 antibody (A03166-2). TOMM40 was detected in an immunocytochemical section of A549 cells. Enzyme anti
M03166 · IFImmunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human osteosarcoma cell line) cells labelin
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000130204-TOMM40/tissue/cerebral+cortex#imid_15462523
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000130204-TOMM40/tissue/colon#imid_15462433
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000130204-TOMM40/tissue/liver#imid_15462450
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000130204-TOMM40/tissue/kidney#imid_15462435
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA036231; green = TOMM40, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA036231; green = TOMM40, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TOMM40 IHC & IF
🧪Run colon first and expect staining in glandular cells (HPA: High in colon glandular cells). Use soft tissue chondrocytes as the biological negative (HPA: Not detected in soft tissue chondrocytes); no cell population in the colon slide is established as an internal negative by the supplied HPA rows, so assess background in noncellular spaces without assuming stromal cells must be unstained.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Soft tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TOMM40 in CACO-2, HEK293, U2OS, HeLa BAC 5698, HeLa , NIH 3T3, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit isotype control, and TOMM40 knockout tissue or a validated peptide-block control (selected A03166-2 caption: rabbit primary antibody; standard IHC controls). Quench endogenous peroxidase before DAB detection and inspect the colon section for pigment or other background that could resemble staining (selected A03166-2 caption: HRP/DAB detection; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative is unreported in the selected A03166-2 paraffin-section caption. That caption reports heat-mediated retrieval in EDTA at pH 8.0 and primary antibody at 2 μg/ml, but does not establish that retrieval is required under other conditions (selected A03166-2 caption). IF/ICC offers a mitochondrial localization cross-check in the listed imaged cell lines (HPA: mitochondria, enhanced), but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; assess colon luminal pigment and nonspecific DAB background when interpreting the tissue stain (standard chromogenic IHC practice).
HPA tissue IHC evidence for TOMM40
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Soft tissue | Chondrocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TOMM40 IHC Tips
Troubleshoot TOMM40 staining in paraffin sections by checking retrieval, compartment, controls, and scoring against the documented tissue pattern.
Which retrieval condition should I try first for TOMM40 in paraffin sections?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet A03166-2). This is the condition documented for the catalog antibody in a paraffin section of human diffuse large B cell lymphoma; the caption does not report a heating duration (datasheet A03166-2). If staining is weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant, and record its buffer, pH, temperature, and duration. Judge improvement by granular cytoplasmic staining (HPA: tissue profile), while checking whether background or tissue damage also increases. Use the documented 2 μg/ml primary incubation as the starting concentration for that comparison (datasheet A03166-2).
How should I assess whether fixation is affecting TOMM40 staining?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not state its fixative (datasheet A03166-2). Record fixative, fixation duration, section thickness, and storage history for each specimen before comparing staining, and keep retrieval and detection consistent across sections. Begin with EDTA pH 8.0 heat retrieval and 2 μg/ml primary antibody incubated overnight at 4°C, as documented for the catalog antibody (datasheet A03166-2). If differently processed specimens disagree, compare matched control tissue processed alongside them and inspect tissue preservation before assigning the difference to TOMM40 abundance. Do not infer a fixation effect from the observed HPA tissue pattern.
What staining pattern supports TOMM40 localisation in tissue sections?
Expect a granular cytoplasmic pattern by chromogenic IHC (HPA: tissue profile), consistent with TOMM40 at the mitochondrial outer membrane (UniProt O96008: subcellular location). At light microscope resolution, individual mitochondrial membranes generally cannot be resolved, so assess the distribution of cytoplasmic granules within intact cells rather than demanding a crisp membrane outline. Compare glandular cells in colon or small intestine, where HPA reports high staining, with neighboring structures on the same section (HPA: colon and small intestine). Predominantly nuclear, uniform extracellular, or diffuse edge restricted DAB should prompt review of retrieval, blocking, and detection. The HPA subcellular record also notes cytosolic localisation, so distribution alone cannot prove mitochondrial specificity (HPA: subcellular).
Could epitope position explain weak TOMM40 staining after retrieval?
TOMM40 has 2 annotated isoforms, but the supplied catalog caption does not map the antibody epitope or establish isoform recognition (UniProt O96008: isoforms; datasheet A03166-2). Its topology places residues 1–76 on the cytoplasmic side and lists 19 membrane spanning segments, with short intermembrane space regions (UniProt O96008: topology). Those features make epitope accessibility a useful troubleshooting question, but they do not identify why a particular section stains weakly. First repeat the documented EDTA pH 8.0 retrieval with matched controls (datasheet A03166-2); then compare any alternative condition on adjacent sections. Avoid assigning an isoform from DAB intensity unless the antibody's epitope and isoform reactivity are established.
How can IF help assess TOMM40 staining alongside a cell identity marker?
Use IF as a complementary localisation check: HPA reports mainly mitochondrial and additional cytosolic TOMM40 signal in cell images (HPA: subcellular). For a glandular epithelial region such as colon, multiplex with an epithelial identity marker and compare TOMM40 signal within marker positive cells (HPA: colon glandular cells, high). Choose spectrally separated fluorophores and consider a far red channel if tissue autofluorescence obscures shorter wavelengths; include single stain and no primary controls when setting exposure and bleed through. Permeabilisation should be matched to epitope location: residues 1–76 face the cytoplasm, while other listed regions face the intermembrane space (UniProt O96008: topology). Because the catalog antibody's epitope is unspecified, test permeabilisation conditions rather than assuming which side it recognizes.
What should I check when TOMM40 DAB staining is widespread or hazy?
The catalog example used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase conjugated secondary for 30 minutes at 37°C (datasheet A03166-2). If staining is hazy, compare a no primary section and review blocking, washing, antibody concentration, and DAB development under the same exposure conditions. Include a peroxidase blocking step and check for residual endogenous enzyme signal; these are general chromogenic IHC controls, not TOMM40 specific findings. Inspect section edges, folds, and damaged areas separately from intact cells before scoring. Compare any residual signal with the reported granular cytoplasmic pattern (HPA: tissue profile).
How should I score TOMM40 IHC across samples with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring because HPA reports high staining in several glandular or epithelial populations and lower staining in macrophages, adipocytes, myocytes, and smooth muscle cells (HPA: tissue IHC). Within a consistent cell compartment, record percentage positive cells and intensity to calculate an H-score, or measure positive cell density per mm² if cell counts are the endpoint. Normalise to the number of evaluable cells or intact tissue area in that compartment, and apply one threshold and DAB development setting across batches. Exclude necrosis, folds, and section edges from the denominator. Report cytoplasmic granularity separately from overall DAB intensity (HPA: tissue profile).
How do I distinguish convincing TOMM40 positivity from staining artefact?
Convincing signal should be granular and predominantly cytoplasmic in intact cells (HPA: tissue profile), in keeping with mitochondrial outer membrane TOMM40 (UniProt O96008: subcellular location). Check whether the stained cell population fits the specimen: HPA reports high signal in colon glandular cells but low signal in lung macrophages and no detected signal in soft tissue chondrocytes (HPA: tissue IHC). Treat predominantly nuclear or extracellular DAB, staining concentrated at section edges, and signal over necrosis as reasons to inspect artefact and tissue preservation. A no primary control helps reveal detection background, including residual endogenous peroxidase; compare controls processed with the same DAB development time. Neither staining intensity nor a single section establishes mitochondrial function.
Boster reagents
Best TOMM40 / Mitochondrial import receptor subunit TOM40 homolog IHC Antibodies
The catalog includes human-reactive TOMM40 antibodies with IHC images from paraffin tissue sections and IF images from cultured cells and a paraffin tissue section (catalog image captions).
Real IHC data
Anti-TOMM40 Antibody ®
Cat # A03166-2
Real IF data
Anti-TOMM40 Antibody (N-Term)
Cat # M03166
A03166-2 has IHC images from human paraffin sections of diffuse large B cell lymphoma, liver cancer, lung adenocarcinoma, and ovarian cancer, plus IF images from A549 cells and a colon cancer section (A03166-2 image captions). M03166 has IF data from U-2 OS cells and lists no IHC application (M03166 image caption; catalog applications).
Which to pick: Choose A03166-2 for human paraffin-section IHC: its own caption documents EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody, but does not report the fixative (A03166-2 IHC caption). For IF/ICC, A03166-2 lists both applications and has A549 cell IF data; M03166 lists IF and shows mitochondrial staining in U-2 OS cells, but does not list ICC (catalog applications; A03166-2 and M03166 IF captions). Neither SKU has documented cross-species reactivity; both list human reactivity, and M03166 is described as polyclonal while A03166-2 has no clonality specified (catalog reactivity; M03166 dilution notes; A03166-2 catalog entry).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O96008 (TOM40_HUMAN, Mitochondrial import receptor subunit TOM40 homolog).
- Human Protein Atlas. TOMM40 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. TOMM40 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the cytosol..
- Human Protein Atlas. TOMM40 antibody validation summary (2 antibodies).
- Comparison of BMIPP-SPECT/CT to (18)FDG-PET/CT for Imaging Brown or Browning Fat in a Preclinical Model. International journal of molecular sciences 2022 — PMC9101718.
- TOMM40/FADS2 Expression Ratio Predicts the Sensitivity to mTOR Inhibitors in Triple-Negative Breast Cancer. Cancer medicine 2026 — PMC13380028.
- Targeting TOMM40 unleashes immunogenic mitophagy to facilitate antigen presentation and immunotherapy sensitization. Journal for immunotherapy of cancer 2026 — PMC13409098.
- lncRNA NKILA Promotes Warburg Effect and Immune Escape in Intrahepatic Cholangiocarcinoma by Regulating the MTX1/TOMM40 Axis. Mediators of inflammation 2025 — PMC12767443.
- PubMed PMID:10520737 — UniProt-cited evidence.
- PubMed PMID:11745481 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TOMM40 (Mitochondrial import receptor subunit TOM40 homolog). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial outer membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03166-2). Validated IHC antibody: A03166-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Soft tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human diffuse large B cell lymphoma tissue; fixative not specified (datasheet A03166-2); 2. Fixation: Image fixative and duration unreported (datasheet A03166-2); 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 A03166-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03166-2); 8. Primary antibody: Rabbit anti-TOMM40, 2-5 μg/ml (datasheet A03166-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03166-2). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Low macrophage staining may be missed (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Specific regulation is not annotated (UniProt). Evidence: UniProt O96008, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A03166-2, PMC13380028. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TOMM40/immunohistochemistry-ihc-guide.