This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
DIABLO / Diablo IAP-binding mitochondrial protein · IHC design guide
Design Immunohistochemistry for DIABLO
Plan chromogenic DIABLO IHC around granular cytoplasmic tissue staining (HPA tissue IHC). Compare high staining in testis pachytene spermatocytes with undetected staining in adipocytes (HPA tissue IHC), and consider apoptosis-associated cytosolic release when scoring (UniProt).
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) | |
| Staining pattern | General granular cytoplasmic staining (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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03790) | |
| Caveat | Apoptosis can shift DIABLO into the cytosol (UniProt) | |
| Regulation | Widespread expression; highest in testis (UniProt) | |
| Isoform / epitope | 3 isoforms; cleavage at 22 or 56 may affect N-terminal epitopes (UniProt) |
Section 1
Recommended DIABLO IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by published protocols for gastric and colorectal carcinoma (PMC4214500) 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 retrieval rule). Use each article’s stated conditions when reproducing its protocol (PMC4214500; PMC3225885).
Section 2
What Is the Expected DIABLO Staining Pattern?
DIABLO is mainly mitochondrial and can also occur in the cytosol, especially after release during apoptosis (UniProt Q9NR28: subcellular location). In paraffin-section IHC, expect granular cytoplasmic staining in positive cells, including gallbladder and stomach glandular cells, pancreatic endocrine cells, and testicular pachytene spermatocytes (HPA: tissue IHC, Supported). A membrane outline is not expected from its topology (UniProt Q9NR28: no transmembrane segment).
| Granular cytoplasmic staining in the listed glandular, endocrine, or spermatocyte populations. | This matches the reported tissue pattern and high staining in those cell types (HPA: tissue IHC). Granules are consistent with mitochondrial localisation; IHC alone does not prove organelle identity (UniProt Q9NR28: mitochondrion; general IHC practice). |
| Predominantly nuclear staining or a sharp cell-membrane outline, with little cytoplasmic signal. | That distribution conflicts with the reported mitochondrial and cytosolic locations and lack of a transmembrane segment (UniProt Q9NR28). Review controls and staining specificity before scoring it as DIABLO (general IHC practice). |
| Strong staining in adipocytes or soft-tissue fibroblasts while expected positive cells are weak. | HPA reports DIABLO as not detected in those cell populations (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible; compare no-primary and detection controls (general IHC practice). |
| Diffuse colour across cells and surrounding tissue, obscuring cytoplasmic granules. | This does not match HPA’s general granular cytoplasmic pattern (HPA: tissue IHC). Excess background may reflect detection chemistry or inadequate blocking or washing; assess the no-primary control (general IHC practice). |
| No staining in a section containing an expected high-staining cell population. | An absent signal is inconclusive if that population is present: gallbladder or stomach glandular cells, pancreatic endocrine cells, and pachytene spermatocytes are reported high (HPA: tissue IHC). Check assay controls and tissue preservation (general IHC practice). |
💡Expected DIABLO appearanceCall a positive result when the expected cell population shows clear granular cytoplasmic staining, potentially high in HPA-listed positive populations; isolated nuclear, membrane-outline, or control-matched diffuse colour is suspect (HPA: tissue IHC; UniProt Q9NR28: location and topology; general IHC practice).
| Tissue and cell population | Expression varies within tissues: HPA reports high staining in gallbladder and stomach glandular cells, pancreatic endocrine cells, and pachytene spermatocytes, but no detection in adipocytes or soft-tissue fibroblasts (HPA: tissue IHC). Score the identified cell population, not whole-section colour (general IHC practice). |
| Subcellular distribution | DIABLO is mitochondrial and may enter the cytosol during apoptosis in a PARL-dependent manner (UniProt Q9NR28: subcellular location). HPA’s tissue IHC description is granular cytoplasmic; a more diffuse cytoplasmic signal requires context and controls, rather than an automatic apoptosis call (HPA: tissue IHC; general IHC practice). |
| Processing and antibody epitope | UniProt lists chains spanning residues 22–239 and 56–239 (UniProt Q9NR28: processing). Antibody epitope location is not supplied, so these annotations cannot predict staining of either form or justify a particular antigen-retrieval condition (general IHC practice). |
| Isoforms and antibody validation | Three isoforms are listed, with isoform 3 described as ubiquitous (UniProt Q9NR28: isoforms and tissue specificity). HPA rates IHC staining with HPA001825 and CAB003857 Supported; those ratings do not identify which isoforms either antibody detects (HPA: antibody validation). |
| IF/ICC Q: what pattern should be expected? | A: Mitochondria are the supported main location; cytosol is uncertain in HPA ICC-IF, while sperm flagellar centriole, mid piece, principal piece, and end piece locations are approved (HPA: subcellular ICC-IF). Interpret IF/ICC on its separate guide page; no IF protocol is specified here. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in an expected positive cell population. | The result conflicts with reported high staining in the specified cells (HPA: tissue IHC); an assay failure or missing target cells remains possible (general IHC practice). | Confirm the relevant cells are present, inspect the positive control, and review retrieval, primary-antibody dilution, detection, and counterstain settings (general IHC practice). |
| Only nuclei or cell borders stain. | This conflicts with DIABLO’s mitochondrial/cytosolic localisation and lack of a transmembrane segment (UniProt Q9NR28). | Compare with a no-primary control and an expected positive tissue; reassess antibody specificity and detection before assigning DIABLO localisation (general IHC practice). |
| Adipocytes or soft-tissue fibroblasts stain strongly. | Both populations are reported not detected by HPA tissue IHC; cross-reactivity or endogenous detection activity may contribute (HPA: tissue IHC; general IHC practice). | Inspect no-primary and detection controls, then compare staining with an HPA-listed high cell population in the same run (general IHC practice). |
| Granules cannot be distinguished from diffuse background. | The expected tissue pattern is granular cytoplasmic (HPA: tissue IHC); nonspecific antibody binding or detection background can obscure it (general IHC practice). | Check no-primary staining, blocking and wash conditions, and primary-antibody dilution; score only cell-associated signal distinguishable from background (general IHC practice). |
| A broadly cytosolic pattern appears without clear granules. | Cytosolic release can accompany apoptosis (UniProt Q9NR28), but HPA tissue IHC generally reports granular cytoplasm (HPA: tissue IHC). | Review morphology and controls; report the observed compartment without inferring apoptosis from DIABLO staining alone (general IHC practice). |
| Staining differs across cell types in one section. | HPA reports cell-specific levels, including high, medium, low, and not-detected populations (HPA: tissue IHC). | Identify and score each relevant population separately; use the reported cell-specific pattern and run controls to interpret unexpected staining (HPA: tissue IHC; 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 score staining in pachytene spermatocytes (HPA: Testis, pachytene spermatocytes High). Use adipose tissue adipocytes as the negative tissue (HPA: Adipose tissue, adipocytes Not detected); on the testis slide, treat cells outside the scored population as internal negatives only where they lack specific staining, since HPA does not designate those cells negative (HPA: Testis, pachytene spermatocytes High).
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 control; an isotype control matched to the primary antibody’s host species, immunoglobulin class, and monoclonal or polyclonal format; and a biological negative such as DIABLO knockout tissue or peptide blocking when the immunizing peptide is available (standard IHC practice). For chromogenic testis sections, block endogenous peroxidase and assess background in the negative controls (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval empirically (standard IHC practice). The fixative is unreported in the selected M03790 paraffin-section caption (M03790 caption: human kidney, paraffin-embedded; fixative not stated). The evidence does not establish whether frozen sections or IF are easier; in testis, distinguish signal in pachytene spermatocytes from differences in cell composition when scoring (HPA: Testis, pachytene spermatocytes High).
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
Troubleshoot DIABLO staining in paraffin sections using its expected granular cytoplasmic pattern, appropriate controls, and consistent scoring (HPA tissue IHC; UniProt Q9NR28 localisation).
How should I retrieve DIABLO in paraffin sections when staining is weak?
Start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Let sections cool consistently, then compare a retrieval-treated section with a matched section processed without retrieval to judge whether the step improves the expected granular cytoplasmic signal (HPA tissue IHC; standard IHC practice). If staining remains weak, optimise heating and primary-antibody concentration separately, keeping detection and exposure conditions constant (standard IHC practice). Use the catalog antibody’s paraffin-section kidney image as a visual reference, while recognising that its caption does not state the fixative (catalog image: M03790).
Can fixation explain weak or diffuse DIABLO staining in paraffin sections?
Target-specific sensitivity of DIABLO staining to fixation is unknown from the supplied evidence; the catalog kidney caption identifies paraffin sections but gives no fixative (catalog image: M03790). Record the actual fixative, fixation duration, tissue thickness, and processing history for each specimen before changing the staining protocol (standard IHC practice). Compare sections from similarly handled blocks using the same pH 6.0, 95–98 °C, 20 min retrieval and identical antibody detection conditions (page retrieval rule; standard IHC practice). If handling histories differ, test representative blocks side by side and report that limitation when comparing signal intensity (standard IHC practice).
Should DIABLO appear as granules or diffuse cytoplasmic staining?
A granular cytoplasmic pattern is the tissue-IHC expectation, consistent with DIABLO’s main mitochondrial localisation (HPA tissue IHC; HPA subcellular: mitochondria supported). DIABLO can also be released into the cytosol during apoptosis in a PARL-dependent manner, so diffuse cytoplasmic staining needs biological and technical assessment (UniProt Q9NR28 localisation). Compare intact and damaged regions on the same section, and examine morphology before interpreting a shift in pattern (standard IHC practice). If the distribution matters to the conclusion, assess a matched mitochondrial marker on adjacent sections and keep staining and imaging conditions comparable (standard IHC practice).
Can this antibody distinguish DIABLO isoforms or processed protein in tissue?
Do not assign a stained cell to a particular DIABLO isoform without epitope and isoform-specific validation: the record lists 3 isoforms but supplies no antibody epitope (UniProt Q9NR28 isoforms; catalog image: M03790). The annotated protein includes a 239-aa precursor and chains spanning residues 22–239 and 56–239, which may expose different regions to an antibody (UniProt Q9NR28 processing; standard immunostaining principle). Check the catalog antibody’s immunogen or epitope documentation before claiming recognition of a processed form (standard IHC practice). DIABLO has no annotated glycosylation sites or modified residues in the supplied record, but that does not establish equal staining across forms (UniProt Q9NR28 annotations).
How can IF help assess DIABLO localisation alongside tissue IHC?
On a matched IF section, multiplex DIABLO with a marker that identifies the expected cell population and, if testing organelle localisation, a mitochondrial marker (standard IF practice; HPA subcellular: mitochondria supported). Choose spectrally separated fluorophores and place the weaker signal in a channel with lower measured tissue autofluorescence, checking single-stain controls for bleed-through (standard IF practice). DIABLO is mainly mitochondrial and is also reported in cytosol, so optimise permeabilisation to admit antibody to intracellular epitopes without erasing spatial detail (HPA subcellular; standard IF practice). Interpret any diffuse signal against cell morphology and controls before relating it to apoptosis (UniProt Q9NR28 localisation; standard IF practice).
What should I check when DIABLO chromogenic staining is widespread?
Compare the distribution with the reported granular cytoplasmic tissue pattern before calling uniform colour DIABLO positive (HPA tissue IHC). Include a section without primary antibody, use an appropriate peroxidase block before chromogenic detection, and inspect whether colour persists where specific antibody binding should be absent (standard IHC practice). Titrate the primary antibody and detection reagents on matched sections while holding the pH 6.0, 95–98 °C, 20 min retrieval constant (page retrieval rule; standard IHC practice). Also inspect section edges, folds, necrotic areas, and counterstain intensity; record these separately from cellular cytoplasmic signal (standard IHC practice).
How should I score DIABLO staining across paraffin specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, then apply the same segmentation and positivity threshold to every section (standard IHC practice). For cellular intensity, score 0–3 and calculate an H-score from intensity-weighted percentages, yielding 0–300; alternatively report the percentage of positive cells (standard IHC practice). For spatial analyses, report positive-cell density per mm² of eligible tissue and state how edges and necrosis were excluded (standard IHC practice). Normalise to the number of eligible cells or eligible tissue area, as appropriate, and keep retrieval, detection, imaging, and scoring rules consistent (standard IHC practice).
What distinguishes convincing DIABLO staining from artefact or misplaced signal?
Convincing staining should be cellular and predominantly granular within cytoplasm, matching the supported tissue-IHC pattern and mitochondrial localisation (HPA tissue IHC; HPA subcellular: mitochondria supported). Check that positive cells belong to the tissue compartment being scored; HPA reports high staining in testis pachytene spermatocytes and no detection in adipocytes (HPA tissue IHC). Treat isolated nuclear colour, section-edge deposits, folds, and necrotic debris cautiously, and compare them with the section lacking primary antibody (standard IHC practice). Residual endogenous peroxidase can also produce chromogen signal, so verify the peroxidase block before interpreting diffuse colour (standard IHC practice).
Boster reagents
Best DIABLO / Diablo IAP-binding mitochondrial protein IHC Antibodies
Anti-DIABLO antibodies have IHC images from human kidney and human, mouse, and rat testis, plus an IF/ICC image from U20S cells (catalog image captions).
Real IHC data
Anti-Smac/Diablo Rabbit Monoclonal Antibody
Cat # M03790
M03790 will render with an IHC image of a paraffin-embedded human kidney section; IF/ICC is listed as an application, but no IF image is supplied (M03790 catalog). PB9860 has IHC images of paraffin-embedded human, mouse, and rat testis and an IF/ICC image of U20S cells (PB9860 catalog image captions).
Which to pick: Choose M03790 for the pictured human kidney paraffin-section IHC example; its caption does not report the fixative (M03790 IHC caption). Choose PB9860 when an IF/ICC image is useful, since its catalog includes one from U20S cells (PB9860 IF caption). For IHC examples across species, PB9860 has pictured human, mouse, and rat testis sections, while M03790 is a rabbit monoclonal with listed human, mouse, and rat reactivity and a pictured human kidney section (PB9860 IHC captions; M03790 catalog and IHC caption).
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). 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03790); Caveat: Apoptosis can shift DIABLO into the cytosol (UniProt); Isoform / epitope: 3 isoforms; cleavage at 22 or 56 may affect N-terminal epitopes (UniProt); Regulation: Widespread expression; highest 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, PMC3225885. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DIABLO/immunohistochemistry-ihc-guide.