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BNIP3 / BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 · IHC design guide
Design Immunohistochemistry for BNIP3
Plan BNIP3 paraffin IHC around cytoplasmic staining in most tissues, with high staining in pancreatic exocrine glandular cells (HPA tissue IHC). The catalog antibody lists an IHC dilution range of 1:50–1:200 (datasheet: M01469); mitochondrial outer membrane localization is a molecular expectation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BNIP3 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 | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in most tissues; high in pancreatic exocrine cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | PPTC7 promotes BNIP3 degradation (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitope side is unmapped (UniProt) |
Section 1
Recommended BNIP3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with four published BNIP3 IHC protocols (PMC5463433; PMC5602480; PMC6070000; PMC3654981).
| Sample | Paraffin-embedded Human pancreatic cancer tissue; fixative not specified (datasheet M01469) |
| Fixation | Image fixative and duration unreported (datasheet M01469); 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 AAEI-2) anti-BNIP3, 1:50-1:200 (datasheet M01469) |
| 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 | BNIP3-positive staining in cells in granular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); evaluate the acetic acid retrieval reported in PMC3654981 if needed.
Section 2
What Is the Expected BNIP3 Staining Pattern?
BNIP3 is a mitochondrial outer membrane protein with a transmembrane segment at residues 164–184 (UniProt Q12983 topology). In paraffin section IHC, expect predominantly cytoplasmic staining, including strong staining in pancreatic exocrine glandular cells and kidney collecting ducts (HPA: cytoplasmic expression in most tissues; High in these cells). HPA rates its tissue IHC pattern Supported, with medium consistency between staining and RNA data (HPA: reliability).
| Granular or punctate cytoplasmic signal in pancreatic exocrine cells or kidney collecting ducts. | This fits BNIP3's mitochondrial outer membrane location and HPA's cytoplasmic IHC pattern; both cell groups are rated High (UniProt Q12983 subcellular location; HPA: High in both). IHC alone does not prove that each colored granule is a mitochondrion. |
| Predominantly nuclear staining, with little cytoplasmic signal, across the section. | Review as a possible nonspecific pattern: HPA reports cytoplasmic IHC staining and UniProt places BNIP3 at mitochondria (HPA: tissue profile; UniProt Q12983 location). UniProt reports a nuclear-envelope shift with EIB 19-kDa coexpression, so that specific context needs separate assessment (UniProt Q12983 location note). |
| Strong staining in cells expected to have low BNIP3 staining, while nearby cells show little signal. | Consider antibody cross-reactivity or endogenous detection activity, then check cell identity and controls (general IHC practice). HPA reports low staining in thyroid glandular cells and splenic red-pulp cells, for example; low is not an absolute negative (HPA: Low in those cells). |
| Diffuse color across cells, stroma and blank areas, obscuring cell boundaries. | Treat the distribution as background until controls clarify it; it cannot establish BNIP3 localisation (general IHC practice). Compare with the expected cytoplasmic, cell-associated pattern and inspect a primary-antibody omission control (HPA: tissue profile; general IHC practice). |
| No convincing signal in pancreatic exocrine glandular cells or kidney collecting ducts. | Those are useful positive-reference cells because HPA rates them High (HPA: High in both). Check tissue preservation, retrieval and detection controls before concluding the sample lacks BNIP3; the HPA rating does not guarantee staining with every antibody or preparation (general IHC practice). |
💡Expected BNIP3 appearanceA convincing positive shows strong, cell-associated cytoplasmic staining in pancreatic exocrine cells or kidney collecting ducts (HPA: High in both; cytoplasmic profile); widespread nuclear color or diffuse stain outside cells warrants a control check (general IHC practice).
| Subcellular location and epitope | BNIP3 sits at the mitochondrial outer membrane, with a transmembrane segment at 164–184 (UniProt Q12983). The supplied sources do not locate the catalog antibody's epitope; topology alone cannot select a retrieval condition or predict epitope accessibility. |
| Choice of positive-reference cells | HPA rates pancreatic exocrine cells, kidney collecting ducts, prostate glandular cells, Leydig cells and selected neurons High (HPA: tissue IHC). Choose an identifiable cell population when judging a section; HPA's low-staining groups are weaker references, not proven negatives (HPA: tissue IHC). |
| Strength of tissue evidence | The HPA tissue profile is Supported, with medium consistency between antibody staining and RNA expression (HPA: reliability). HPA also lists two IHC antibodies as Supported (HPA: HPA003015 and CAB011676); those ratings do not independently validate a different catalog antibody. |
| Retrieval and detection | The supplied UniProt and HPA records give no BNIP3-specific retrieval setting or fixation-sensitivity result. Follow the IHC-validated antibody's supplied IHC-P procedure where available, and use routine assay controls to assess retrieval and detection (general IHC practice). |
| Q: Does the IHC pattern establish an IF/ICC result? | A: No. HPA's subcellular summary says Membrane, but provides no main location or ICC-IF image-bearing cell lines here (HPA: subcellular record). Use the separate IF/ICC guide for that application; the paraffin IHC observations do not establish an IF staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-reference tissue is blank. | The assay may have failed, or this preparation or antibody may not reveal detectable signal (general IHC practice). HPA rates pancreatic exocrine cells and kidney collecting ducts High, but does not validate every assay condition (HPA: tissue IHC). | Confirm the expected cells are present; check the IHC-validated antibody's IHC-P procedure, retrieval step and detection control before interpreting a negative result (general IHC practice). |
| Signal appears mainly in nuclei. | A widespread nuclear pattern conflicts with HPA's cytoplasmic tissue profile and UniProt's mitochondrial location (HPA: tissue profile; UniProt Q12983). The EIB 19-kDa coexpression note is a specific exception (UniProt Q12983). | Compare with a positive-reference section and a primary-antibody omission control; investigate the exceptional context only if relevant to the sample (general IHC practice; UniProt Q12983 location note). |
| Brown color fills stroma or covers the section uniformly. | Excess background or endogenous detection activity can obscure a cell-associated IHC signal (general IHC practice); that distribution does not match HPA's cytoplasmic cellular profile (HPA: tissue IHC). | Inspect an omission control and the detection reagents; adjust blocking, washes or chromogen development according to the assay controls (general IHC practice). |
| A low-staining cell group looks as strong as the positive reference. | Cross-reactivity or background is possible, but HPA's Low category does not mean zero protein (HPA: low-staining cell groups; general IHC practice). Cell identity and local staining distribution also matter. | Compare the same run with a HPA High reference, review morphology and omission-control signal, then assess whether the unexpected stain is cell-associated (HPA: tissue IHC; general IHC practice). |
| A different antibody gives a different distribution. | HPA reports Supported IHC staining for HPA003015 and CAB011676, without establishing equivalence to the catalog antibody (HPA: antibody validation). Differences may reflect assay conditions or antibody specificity (general IHC practice). | Run comparable sections with matched detection controls and assess the expected cells and compartment; follow each antibody's own validated IHC-P instructions (general IHC practice). |
| Punctate signal is hard to distinguish from background. | BNIP3's mitochondrial location supports a cytoplasmic expectation, but individual puncta cannot be assigned to mitochondria by chromogenic IHC alone (UniProt Q12983 location; HPA: tissue profile). | First confirm cell-associated staining in a HPA High population and check the omission control; reserve organelle colocalisation questions for the separate IF/ICC design (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing BNIP3 staining
M01469 · IHCImmunohistochemical analysis of paraffin-embedded Human pancreatic cancer, using the Antibody at 1:100 dilution.
M01469 · IHCImmunohistochemical analysis of paraffin-embedded Human placenta, using the Antibody at 1:100 dilution.
M01469 · IFImmunofluorescent analysis using the Antibody at 1:500 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000176171-BNIP3/tissue/cerebral+cortex#imid_987392
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000176171-BNIP3/tissue/colon#imid_987318
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000176171-BNIP3/tissue/liver#imid_987311
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000176171-BNIP3/tissue/pancreas#imid_987278
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BNIP3 IHC & IF
🧪Run kidney first: collecting duct cells should stain strongly (HPA: High in kidney collecting ducts). HPA detects BNIP3 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect cells judged unstained on the positive slide to retain counterstain without specific chromogen (HPA: no negative tissue rows).
Positive control tissue: Cerebellum (Cells in granular layer, HPA High)
Negative control tissue: None in HPA: BNIP3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for BNIP3; derive a cell-line control from the positive tissue's cell type (Cells in granular layer) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only section; a nonimmune control matched to the primary antibody’s host species and immunoglobulin type; and a BNIP3 knockout specimen as a biological negative. Block endogenous peroxidase for chromogenic IHC, and check kidney sections for endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected M01469 paraffin-section caption does not report a fixative (M01469 caption: fixative unreported). Antigen retrieval dependence is unreported, so assess retrieval with the catalog antibody’s IHC-P protocol and verify the expected collecting duct staining (HPA: High in kidney collecting ducts). Neither frozen sections nor IF is established as easier by the supplied evidence; kidney sections can show endogenous biotin signal with biotin-based detection (standard IHC practice).
HPA tissue IHC evidence for BNIP3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: BNIP3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced BNIP3 IHC Tips
Troubleshoot BNIP3 staining in paraffin sections by checking retrieval, compartment, controls, and scoring before interpreting changes in chromogenic signal.
What retrieval should I start with for BNIP3 in paraffin sections?
Start with citrate buffer at pH 6.0 for heat retrieval at 95–98 °C for 20 min (page retrieval setting). Allow sections to cool in buffer, then compare staining with a matched section processed identically except for retrieval (standard IHC practice). If staining is weak, vary heating time in small steps while keeping antibody dilution and detection constant; excessive heating can damage morphology (standard IHC practice). Assess cytoplasmic signal in pancreatic exocrine glandular cells as a reference, while treating the selected pancreatic cancer image at 1:100 as evidence of staining in paraffin sections only (HPA: High in pancreatic exocrine glandular cells; M01469 caption: paraffin section, 1:100).
Could fixation explain weak or uneven BNIP3 staining?
BNIP3-specific fixation sensitivity is unknown from the supplied evidence; the selected paraffin-section caption does not state its fixative (M01469 caption: fixative not stated). Record the actual fixative and fixation duration for each specimen, and compare sections processed under the same conditions before changing antibody concentration (standard IHC practice). If staining varies across a batch, inspect morphology and section adhesion, then repeat retrieval and detection on matched sections to separate processing variation from staining variation (standard IHC practice). Do not infer a BNIP3 fixation effect from its mitochondrial outer-membrane topology or annotated phosphoserines, because neither establishes fixation sensitivity (UniProt Q12983: topology and modified residues).
Where should BNIP3 staining appear, and how should nuclear signal be handled?
Expect predominantly cytoplasmic chromogenic staining in tissue sections, consistent with the tissue staining profile, while BNIP3 is assigned to mitochondria and their outer membrane (HPA: cytoplasmic expression in most tissues; UniProt Q12983: subcellular location). A granular cytoplasmic pattern may fit that location, but chromogenic staining alone does not establish mitochondrial colocalisation (UniProt Q12983: mitochondrial location; standard IHC interpretation). Review nuclear or nuclear-envelope staining separately: a nuclear-envelope shift is described with EIB 19-kDa protein coexpression, which the tissue caption does not establish (UniProt Q12983: coexpression-dependent shift; M01469 caption). Compare suspicious compartments with a negative control and preserved morphology before assigning them to BNIP3 (standard IHC practice).
How can epitope placement affect interpretation of BNIP3 staining?
Check the antibody's stated immunogen or epitope before changing retrieval or permeabilisation; its position is not supplied here (supplied antibody evidence: epitope unspecified). BNIP3 has an annotated transmembrane segment at residues 164–184 and a single annotated chain spanning residues 1–194 (UniProt Q12983: topology and processing). Phosphoserines are annotated at residues 54, 66, 86, 92, and 95, so a defined epitope near those sites warrants careful comparison across processing conditions without assuming phosphorylation changes antibody binding (UniProt Q12983: modified residues; standard IHC interpretation). No isoforms are annotated in the supplied record; do not attribute differing tissue patterns to a specific isoform without independent evidence (UniProt Q12983: isoforms).
How can IF help resolve ambiguous chromogenic BNIP3 staining?
Use IF on a matched section to examine whether BNIP3 signal overlaps an independently validated marker of the expected cell type, such as pancreatic exocrine glandular cells when evaluating pancreas (HPA: High in pancreatic exocrine glandular cells; standard IF practice). Select fluorophores after measuring tissue autofluorescence in an unstained section, and favor a channel with a clear signal above that background (standard IF practice). Because the antibody epitope is unspecified, choose and titrate permeabilisation only after establishing whether the epitope lies on an accessible or membrane-protected side (supplied antibody evidence: epitope unspecified; UniProt Q12983: outer-membrane protein). Keep IF findings separate from the selected antibody's paraffin-section IHC evidence at 1:100 (M01469 caption).
What should I change when BNIP3 chromogenic staining has high background?
Compare a section without primary antibody with the stained section to identify signal arising from detection reagents or endogenous tissue activity (standard chromogenic IHC practice). Apply a peroxidase block for an enzyme-based detection workflow, then check whether DAB develops in the control before adjusting the primary antibody (standard chromogenic IHC practice). If diffuse background persists, titrate around the documented 1:100 dilution for the selected paraffin-section image while holding retrieval, incubation, and development time constant (M01469 caption: 1:100; standard IHC optimisation). Judge improvement against cytoplasmic staining and tissue morphology, rather than accepting reduced overall colour as evidence of specificity (HPA: cytoplasmic tissue profile; standard IHC interpretation).
How should I score BNIP3 across sections with variable cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring; pancreatic exocrine glandular cells have reported high staining and can serve as a tissue reference (HPA: High in pancreatic exocrine glandular cells; standard IHC scoring practice). For cellular staining, report the percentage of positive cells and an H-score based on the fractions at each intensity; keep the intensity thresholds fixed across slides (standard IHC scoring practice). Normalise counts to the number of evaluable cells in the chosen compartment, or report positive-cell density per mm² of viable annotated area when cellularity differs (standard IHC scoring practice). Exclude necrotic and poorly preserved regions using the same criteria for every specimen, and record batch and retrieval conditions alongside scores (standard IHC scoring practice).
How can I distinguish genuine BNIP3 signal from section artefacts?
Give greatest weight to staining in the expected cytoplasmic compartment and an appropriate cell population; BNIP3 is mitochondrial, and pancreatic exocrine glandular cells show high tissue staining (UniProt Q12983: location; HPA: tissue profile). Treat isolated nuclear signal cautiously unless the relevant EIB 19-kDa coexpression context is independently established (UniProt Q12983: coexpression-dependent nuclear-envelope shift). Inspect section edges and necrotic areas for staining that tracks tissue damage, and compare an omission control for endogenous enzyme or detection signal (standard chromogenic IHC practice). HPA rates tissue staining as Supported with medium agreement to RNA data, so corroborate unexpected compartment or cell-type patterns independently (HPA: reliability description; standard IHC interpretation).
Boster reagents
Best BNIP3 / BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 IHC Antibodies
M01469 has human paraffin-section IHC images and an IF image (catalog image captions); both catalog antibodies list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-BNIP3 Rabbit Monoclonal Antibody
Cat # M01469
M01469 is the sole rendered card; its IHC captions show human pancreatic cancer, placenta, thyroid cancer and kidney in paraffin sections, while its IF caption gives no sample type (catalog image captions). M01469-2 lists IHC and IF/ICC applications and human, mouse and rat reactivity, but has no supplied IHC or IF image captions and will not render as a card (catalog applications, reactivity and image captions; cards payload).
Which to pick: Choose M01469 for tissue IHC because its own images document human paraffin sections; the fixative is unreported (M01469 IHC image captions). Choose M01469 for IF/ICC when image evidence matters: it lists both applications and has an IF image, while M01469-2 lists both applications without a supplied image (catalog applications and image captions). Both are rabbit monoclonals listed as reactive with human, mouse and rat, so the supplied evidence does not establish a cross-species advantage for either SKU (catalog host, clone and reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q12983 (BNIP3_HUMAN, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3).
- Human Protein Atlas. BNIP3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. BNIP3 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. BNIP3 antibody validation summary (2 antibodies).
- Chemoresistance of colorectal cancer to 5-fluorouracil is associated with silencing of the BNIP3 gene through aberrant methylation. Journal of Cancer 2017 — PMC5463433.
- Expression and Functional Characterization of the BNIP3 Protein in Renal Cell Carcinomas. Translational oncology 2017 — PMC5602480.
- Upregulation of BCL2 19 kD Protein-Interacting Protein 3 (BNIP3) is Predictive of Unfavorable Prognosis in Uveal Melanoma. Medical science monitor : international medical journal of experimental and clinical research 2018 — PMC6070000.
- Peroxisome proliferator-activated receptor δ agonist, HPP593, prevents renal necrosis under chronic ischemia. PloS one 2013 — PMC3654981.
- PubMed PMID:7954800 — UniProt-cited evidence.
- PubMed PMID:9396766 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M01469 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in granular layer); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human pancreatic cancer tissue; fixative not specified (datasheet M01469); 2. Fixation: Image fixative and duration unreported (datasheet M01469); 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 AAEI-2) anti-BNIP3, 1:50-1:200 (datasheet M01469); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; epitope side is unmapped (UniProt); Regulation: PPTC7 promotes BNIP3 degradation (UniProt). Evidence: UniProt Q12983, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet M01469, PMC5463433, PMC5602480. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BNIP3/immunohistochemistry-ihc-guide.