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DIABLO / Diablo IAP-binding mitochondrial protein · IHC design guide
Design Immunohistochemistry for DIABLO
Plan paraffin IHC for DIABLO using its granular cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation, controls and scoring, with testis pachytene spermatocytes as a high staining reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DIABLO 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); mitochondria (UniProt) | |
| Staining pattern | Granular cytoplasm; high in testis pachytene spermatocytes (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Gallbladder+4 more · see all | |
| Negative control | Adipose tissue+1 more · see all |
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 | Apoptosis can release DIABLO into the cytosol (UniProt) | |
| Regulation | Highest expression in testis (UniProt) | |
| Isoform / epitope | 3 isoforms; 22–239 and 56–239 forms warrant epitope review (UniProt) |
Section 1
Recommended DIABLO IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published DIABLO methods for gastrointestinal carcinoma (PMC4214500), rat endometrium (PMC194660), and cervical cancer (PMC3225885).
| Sample | Paraffin-embedded human kidney tissue; fixative not specified (datasheet M03790) |
| Fixation | Image fixative and duration unreported (datasheet M03790); 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 monoclonal (clone FGG-4) anti-DIABLO, 1:50 (datasheet M03790) |
| 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 | DIABLO-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen-retrieval setting); the published methods use their own retrieval conditions (PMC4214500; PMC194660; PMC3225885).
Section 2
What Is the Expected DIABLO Staining Pattern?
DIABLO should appear mainly as granular cytoplasmic staining consistent with mitochondria in expressing cells (HPA tissue IHC; HPA subcellular: supported mitochondrial location). Strong examples include gallbladder and stomach glandular cells, pancreatic endocrine cells, and testis pachytene spermatocytes (HPA: High). Cytosolic DIABLO can occur during apoptosis (UniProt Q9NR28: apoptosis-dependent release). DIABLO has no transmembrane segment (UniProt Q9NR28 topology). The HPA tissue IHC pattern is Supported, with high consistency between staining and RNA data (HPA tissue IHC).
| Granular cytoplasmic staining in gallbladder or stomach glandular cells, pancreatic endocrine cells, or pachytene spermatocytes (HPA: High). | This matches the reported tissue pattern and predominant mitochondrial location (HPA tissue IHC; HPA subcellular: supported). Judge intensity within the identified cell population: neighboring cells need not share its staining level (HPA: cell-specific tissue observations). |
| Predominantly nuclear staining, with little granular cytoplasmic signal, in an otherwise positive region. | Nuclear localization is outside the reported principal pattern (HPA tissue IHC; UniProt Q9NR28 subcellular location). Treat it as suspect and compare controls before scoring it as DIABLO; nonspecific antibody binding or detection artefact is possible (general IHC practice). |
| Strong staining in adipocytes or soft-tissue fibroblasts while expected positive cells are also present. | HPA reports DIABLO as Not detected in those respective cell populations (HPA: adipocytes; soft-tissue fibroblasts). Check cell identification and negative controls: cross-reactivity or endogenous detection activity may explain discordant staining (general IHC practice). |
| Diffuse color over tissue, stroma, and blank areas, obscuring cell boundaries and granules. | This does not resolve the reported granular cytoplasmic pattern (HPA tissue IHC). Broad deposition can reflect background from blocking, detection, or washing steps; inspect a reagent control and distinguish precipitate from cellular staining (general IHC practice). |
| No convincing signal in a gallbladder, stomach, pancreas, or testis cell population reported as High (HPA tissue IHC). | An absent result conflicts with those HPA observations but does not alone prove biological absence. Confirm the sampled cell population, then assess retrieval, primary antibody, detection, and counterstain settings against a working control (general IHC practice). |
💡Expected DIABLO appearanceCall a positive result when identified HPA High cells show clear, granular cytoplasmic staining consistent with mitochondrial DIABLO (HPA tissue IHC; HPA subcellular); isolated nuclear color or uniform deposit across unrelated cells is suspect (general IHC practice).
| Cell population and tissue selection | HPA reports High staining in selected glandular cells, pancreatic endocrine cells, and pachytene spermatocytes, but Not detected in adipocytes and soft-tissue fibroblasts (HPA tissue IHC). Use the named cells when judging a control; a whole-section average can hide a focal positive population (general IHC practice). |
| Compartment and apoptosis | The main location is mitochondrial, with a granular cytoplasmic tissue pattern (HPA subcellular: supported; HPA tissue IHC). DIABLO may enter the cytosol when cells undergo apoptosis (UniProt Q9NR28). A less punctate cytoplasmic pattern therefore needs tissue context; it is not, by itself, evidence of failed staining. |
| Protein forms and antibody recognition | UniProt lists three isoforms and chains beginning at residues 22 and 56 (UniProt Q9NR28 processing). Antibody epitope information is absent from this payload, so these annotations cannot establish which forms the IHC-validated antibody detects. Interpret a negative section using a positive tissue control rather than assuming loss of one form (general IHC practice). |
| Antibody validation and IF/ICC Q&A | Q: Should IF/ICC show the same main compartment? A: Mitochondria are the supported main location; cytosol is an uncertain additional ICC-IF location (HPA subcellular). HPA001825 is Supported for both IHC and ICC; CAB003857 is Supported for IHC, while CAB016688 is Supported for ICC (HPA antibodies). IF/ICC protocol choices belong to its own guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported High cell population is unstained, although the section has intact morphology (HPA tissue IHC). | The expected cells may be absent from the sampled area, or a routine retrieval, antibody, or detection step may have failed (general IHC practice). | Verify the cell population and run a known positive section through the same IHC workflow; review retrieval and reagent records before interpreting the negative result (general IHC practice). |
| Signal is pale and lacks the expected granules in otherwise positive cells (HPA tissue IHC). | Weak detection or excessive counterstain can obscure fine cytoplasmic detail (general IHC practice). | Compare a working positive control and counterstain balance, then adjust one routine IHC variable at a time; no DIABLO-specific dilution or retrieval condition is supplied here (general IHC practice). |
| Nuclei dominate the stain while cytoplasm is weak. | A nuclear dominant pattern conflicts with the recorded mitochondrial and cytosolic locations (HPA subcellular; UniProt Q9NR28); nonspecific signal is possible (general IHC practice). | Inspect the no-primary control and repeat with an IHC-supported antibody if available (HPA antibodies; general IHC practice). Score DIABLO only where the cellular pattern is credible. |
| Adipocytes or soft-tissue fibroblasts stain strongly (HPA: Not detected). | Cell misidentification, antibody cross-reactivity, or endogenous detection activity could create a misleading positive (general IHC practice). | Confirm the cell type on the counterstained section and compare appropriate negative controls; check detection blocking if color persists without primary antibody (general IHC practice). |
| Brown color coats many cell types and extracellular areas without discrete granules. | This departs from the granular cytoplasmic HPA profile (HPA tissue IHC) and may reflect chromogen deposit or broad background (general IHC practice). | Inspect blank areas and a no-primary control, then review washing, blocking, and chromogen handling; avoid assigning diffuse deposit to DIABLO-positive cells (general IHC practice). |
| Staining becomes more diffuse within cytoplasm in a region with apoptotic morphology. | DIABLO can be released from mitochondria into cytosol during apoptosis (UniProt Q9NR28); HPA lists cytosol as an uncertain additional ICC-IF location (HPA subcellular). | Record the morphology and compare adjacent viable cells and controls. Do not call apoptosis from DIABLO staining alone; assess the pattern alongside independent morphological evidence (general IHC practice). |
Experiment evidence
IHC & IF images showing DIABLO staining
M03790 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using Smac/Diablo Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000184047-DIABLO/tissue/cerebral+cortex#imid_1321580
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000184047-DIABLO/tissue/colon#imid_1321498
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000184047-DIABLO/tissue/liver#imid_1321507
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000184047-DIABLO/tissue/kidney#imid_1321500
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB016688; green = DIABLO, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB016688; green = DIABLO, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DIABLO IHC & IF
🧪Run testis first and look for staining in pachytene spermatocytes (HPA: High in testis pachytene spermatocytes; UniProt: highest tissue expression in testis). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the testis slide, adjacent stromal fibroblasts should have little staining relative to spermatocytes, but treat them as a background comparator rather than a confirmed DIABLO-negative population (HPA: soft tissue fibroblasts Not detected).
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DIABLO in A-431, U-251MG, U2OS, Sperm, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use a matched DIABLO knockout specimen or peptide competition, if available, to assess specificity (standard IHC practice). For chromogenic detection, block endogenous peroxidase and assess residual testis background on the no-primary section (standard IHC practice).
⚠️Feasibility: Paraffin-section IHC is documented for the selected antibody in human kidney, but its caption does not report a fixative (M03790 tissue-IHC caption: paraffin-embedded human kidney; fixative unreported). A DIABLO-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence, and the ICC-IF images do not establish whether IF or frozen sections are easier than paraffin IHC (HPA: ICC-IF images in A-431, U-251MG, U2OS and sperm). In testis, distinguish true cellular staining from residual chromogenic background using the no-primary control (HPA: High in pachytene spermatocytes; standard IHC practice).
HPA tissue IHC evidence for DIABLO
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 |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Pancreas | Pancreatic endocrine cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced DIABLO IHC Tips
Use these checks to optimise DIABLO staining in paraffin sections and distinguish its expected granular cytoplasmic signal from artefact.
Which retrieval conditions should I use if DIABLO staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). If staining remains weak, compare a shorter heating time on adjacent sections while keeping antibody concentration and detection conditions fixed (standard IHC optimisation practice). Allow sections to cool consistently before staining, since differences in cooling can complicate comparisons (standard IHC practice). Score granular cytoplasmic signal in the same cell population across conditions; DIABLO is mainly mitochondrial, so stronger diffuse staining alone does not establish better retrieval (HPA: general granular cytoplasmic tissue pattern; HPA: mitochondrial location supported).
Could fixation explain weak or uneven DIABLO staining?
The selected M03790 image identifies paraffin-embedded human kidney but does not state its fixative, so DIABLO-specific fixation sensitivity is unknown (M03790 caption). Record the fixative, fixation duration and processing history for each specimen before comparing staining intensities (standard IHC practice). For a controlled comparison, stain adjacent sections from similarly processed blocks together using the same pH 6.0 citrate retrieval at 95–98 °C for 20 min (page retrieval setting). If a specimen remains weak, report the processing difference as a possible confounder rather than attributing it to a proven DIABLO fixation effect (standard IHC interpretation practice).
What DIABLO pattern should I expect in paraffin sections?
Look first for granular cytoplasmic staining, the supported tissue pattern, consistent with DIABLO’s predominantly mitochondrial localisation (HPA: tissue IHC profile; HPA: mitochondrial location supported). Cytosolic DIABLO can occur after apoptosis, but morphology and a single chromogenic stain cannot establish its release mechanism (UniProt Q9NR28: apoptotic cytosolic release; standard IHC interpretation practice). Compare intact cells within the same section and note whether apparent diffuse staining concentrates near damaged areas (standard IHC interpretation practice). Treat predominantly nuclear or extracellular colour as unexpected, then review counterstain, tissue integrity and a control stained without primary antibody before assigning it to DIABLO (HPA: tissue IHC profile; standard IHC practice).
Can this stain distinguish DIABLO isoforms or processed protein?
Do not assign an isoform from chromogenic staining alone: DIABLO has 3 annotated isoforms, while the supplied M03790 caption gives no epitope or isoform specificity (UniProt Q9NR28: isoforms; M03790 caption). The record describes a 239-aa precursor and chains spanning residues 22–239 and 56–239 (UniProt Q9NR28: processing). Check the antibody’s documented immunogen or epitope before claiming that a stain recognises either processed chain (standard antibody validation practice). If epitope information is unavailable, report the result as DIABLO immunoreactivity and compare its cellular pattern with independent evidence, rather than naming a particular isoform or cleavage state (standard IHC interpretation practice).
How can IF help assess the DIABLO pattern seen by IHC?
On a separate IF section, multiplex DIABLO with a validated marker for the cell population being scored and a mitochondrial marker to assess the expected compartment (HPA: supported mitochondrial location; standard IF practice). Choose spectrally separated fluorophores after checking unstained tissue for autofluorescence, and use matched single-stain controls to assess bleed-through (standard IF practice). Use a mild, consistently applied permeabilisation step so antibodies can reach the intracellular DIABLO epitope, adjusting it only after checking the antibody’s documented epitope and IF suitability (UniProt Q9NR28: mitochondrial and cytosolic localisation; standard IF practice). Report IF colocalisation as supporting spatial evidence, not proof of DIABLO release or antibody specificity (standard IF interpretation practice).
How do I troubleshoot diffuse chromogenic background?
Compare the test section with a section stained without primary antibody to locate background from the detection system (standard IHC practice). In a peroxidase and DAB workflow, apply a peroxidase block and check for residual colour in the control; these are general workflow steps, not DIABLO-specific evidence (standard chromogenic IHC practice). Review blocking, wash consistency, antibody concentration and development time one variable at a time while preserving tissue morphology (standard IHC optimisation practice). Retain a condition only if it improves granular cytoplasmic contrast in intact cells, the supported DIABLO tissue pattern, without increasing diffuse colour (HPA: tissue IHC profile; standard IHC interpretation practice).
How should I score DIABLO across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartments before scoring, and keep section thickness, retrieval and DAB development consistent across specimens (standard IHC quantification practice). An H-score combines the percentage of cells at each intensity grade, typically 0–3, into a 0–300 result; record percentage positive separately when useful (standard IHC scoring practice). For focal staining, report positive-cell density per mm² of viable tissue or the defined region of interest (standard image analysis practice). Normalise comparisons to the same eligible cell population and viable tissue area, excluding folds, edges and necrosis; annotate whether scoring targets granular cytoplasmic signal (HPA: tissue IHC profile; standard IHC quantification practice).
When is a DIABLO-positive area convincing rather than artefactual?
A convincing result shows reproducible granular cytoplasmic signal in intact cells, matching the supported tissue pattern and predominant mitochondrial location (HPA: tissue IHC profile; HPA: mitochondrial location supported). High staining in gallbladder glandular cells and pancreatic endocrine cells offers documented tissue context, while adipocytes were reported as undetected in the HPA dataset (HPA: tissue IHC). Check whether colour instead follows section edges, necrosis or damaged cells, which can distort chromogenic interpretation (standard IHC practice). Unexpected nuclear or extracellular signal, or colour persisting without primary antibody, warrants review of morphology, peroxidase blocking and detection controls before a DIABLO-positive call (HPA: tissue IHC profile; standard IHC practice).
Boster reagents
Best DIABLO / Diablo IAP-binding mitochondrial protein IHC Antibodies
Anti-DIABLO IHC images cover paraffin-embedded human kidney and human, mouse, and rat testis; an IF image shows U20S cells (M03790 IHC image caption; PB9860 IHC and IF image captions).
Real IHC data
Anti-Smac/Diablo Rabbit Monoclonal Antibody
Cat # M03790
M03790 is the sole card that will render, with an IHC image of paraffin-embedded human kidney (M03790 IHC image caption). Its catalog lists IHC and IF/ICC applications and human, mouse, and rat reactivity; no IF image is supplied for this SKU (M03790 catalog).
Which to pick: For tissue IHC, M03790 offers a rabbit monoclonal with a human kidney paraffin-section example; the caption does not report the fixative (M03790 catalog; M03790 IHC image caption). For IF/ICC, PB9860 has an image of U20S cells, while M03790 lists IF/ICC without a supplied IF image (PB9860 IF image caption; M03790 catalog). For cross-species tissue IHC, PB9860 has paraffin-section images from human, mouse, and rat testis, though those captions do not report fixation (PB9860 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NR28 (DBLOH_HUMAN, Diablo IAP-binding mitochondrial protein).
- Human Protein Atlas. DIABLO tissue IHC expression (reliability: Supported).
- Human Protein Atlas. DIABLO subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the cytosol, flagellar centriole, mid piece, principal piece and end piece..
- Human Protein Atlas. DIABLO antibody validation summary (3 antibodies).
- Smac/DIABLO expression in human gastrointestinal carcinoma: Association with clinicopathological parameters and survivin expression. Oncology letters 2014 — PMC4214500.
- Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17beta-estradiol at estrus. Reproductive biology and endocrinology : RB&E 2003 — PMC194660.
- Apoptin causes apoptosis in HepG-2 cells via Ca(2+) imbalance and activation of the mitochondrial apoptotic pathway. Cancer medicine 2023 — PMC10134343.
- High Smac/DIABLO expression is associated with early local recurrence of cervical cancer. BMC cancer 2006 — PMC3225885.
- PubMed PMID:10929711 — UniProt-cited evidence.
- PubMed PMID:10950947 — UniProt-cited evidence.
- PubMed PMID:14523016 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DIABLO (Diablo IAP-binding mitochondrial protein). Expected localisation Granular cytoplasm in tissue (HPA tissue IHC); mitochondria (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M03790 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human kidney tissue; fixative not specified (datasheet M03790); 2. Fixation: Image fixative and duration unreported (datasheet M03790); 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 monoclonal (clone FGG-4) anti-DIABLO, 1:50 (datasheet M03790); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Apoptosis can release DIABLO into the cytosol (UniProt); Isoform / epitope: 3 isoforms; 22–239 and 56–239 forms warrant epitope review (UniProt); Regulation: Highest expression in testis (UniProt). Evidence: UniProt Q9NR28, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet M03790, PMC4214500, PMC194660. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DIABLO/immunohistochemistry-ihc-guide.