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BAD / Bcl2-associated agonist of cell death · IHC design guide
Design Immunohistochemistry for BAD
Plan paraffin-section BAD IHC around widespread cytoplasmic staining (HPA tissue IHC) and a 1:50 catalog-antibody starting dilution (datasheet: M03520 IHC caption). Compare signal across cell types while allowing for phosphorylation-dependent redistribution (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BAD 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 | Cytoplasm (HPA tissue IHC); mitochondrial outer membrane (UniProt) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03520) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Ovary+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03520) | |
| Caveat | Phosphorylation can shift BAD toward the cytoplasm (UniProt) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; one full-length chain (UniProt) |
Section 1
Recommended BAD IHC & IF Protocols
The catalog antibody’s IHC protocol is followed by four published BAD IHC protocols, including total BAD and phospho-BAD staining (PMC3787924; PMC5442111; PMC11366015; PMC4145317).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet M03520) |
| Fixation | Image fixative and duration unreported (datasheet M03520); 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 M03520); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03520) |
| Primary antibody | Rabbit monoclonal (clone CHC-2) anti-BAD, 1:50 (datasheet M03520) |
| Primary incubation | Overnight at 4 °C (datasheet M03520) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M03520) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | BAD-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with the catalog antibody’s heat-mediated EDTA pH 8.0 retrieval (datasheet: M03520); published retrieval conditions belong to their cited protocols (PMC3787924; PMC11366015).
Section 2
What Is the Expected BAD Staining Pattern?
BAD is found at the mitochondrial outer membrane and in the cytoplasm; phosphorylation can shift it toward the cytoplasm (UniProt Q92934 localization; UniProt Q92934 topology: no transmembrane segment). In paraffin IHC, expect cytoplasmic staining in cells such as kidney tubule cells and bronchial respiratory epithelial cells (HPA tissue IHC: High). HPA describes the tissue pattern as ubiquitous cytoplasmic expression, with Approved reliability, medium staining–RNA consistency, and external verification pending (HPA tissue IHC).
| Cytoplasmic staining in kidney tubule cells, bronchial respiratory epithelial cells, or placental trophoblastic cells. | This fits HPA’s High staining calls for those cell types (HPA tissue IHC: High). BAD can also associate with the mitochondrial outer membrane (UniProt Q92934 localization). A chromogenic cytoplasmic signal alone cannot establish precise mitochondrial localization (standard IHC interpretation). |
| Predominantly nuclear staining, with little cytoplasmic signal in a positive control. | A nuclear dominant pattern conflicts with the reported cytoplasmic and mitochondrial locations (UniProt Q92934 localization; HPA tissue IHC: ubiquitous cytoplasmic expression). Treat it as suspect, then compare the staining with an independently validated antibody or an appropriate negative control (standard IHC practice). |
| Strong staining confined to skeletal muscle myocytes, smooth muscle cells, or ovarian stroma cells. | HPA reports BAD as Not detected in those cell types (HPA tissue IHC: Not detected). Such staining warrants checks for cross-reactivity or endogenous detection activity (standard IHC practice). A Not detected call is a reference pattern, not proof that every specimen must be blank (HPA tissue IHC: Approved; external verification pending). |
| Diffuse color over cells and surrounding tissue, without a clear cellular pattern. | Uniform background is difficult to reconcile with HPA’s cell-specific staining calls (HPA tissue IHC). Check blocking, detection-only background, and antibody concentration as general causes of nonspecific chromogenic signal (standard IHC practice); do not score haze as BAD-positive cytoplasm (standard IHC interpretation). |
| No signal in kidney tubule cells or bronchial respiratory epithelial cells used as positive controls. | Both are High reference cell types (HPA tissue IHC: High). First assess section quality, antigen retrieval, primary antibody handling, and detection performance (standard IHC practice). A failed positive control makes a negative result elsewhere uninterpretable; HPA’s Approved assessment does not guarantee staining in every preparation (HPA tissue IHC). |
💡Expected BAD appearanceCall the result positive when defined cytoplasmic staining is visible in HPA High cell types, such as kidney tubule cells or bronchial respiratory epithelial cells (HPA tissue IHC: High; ubiquitous cytoplasmic expression); predominantly nuclear color or strong signal in HPA Not detected cell types is suspect (UniProt Q92934 localization; HPA tissue IHC: Not detected).
| Compartment and phosphorylation state | BAD occupies the mitochondrial outer membrane and cytoplasm; upon phosphorylation it locates to the cytoplasm (UniProt Q92934 localization). A less distinctly mitochondrial appearance need not be an IHC failure, but this record gives no threshold for scoring that shift in paraffin sections (UniProt Q92934 localization). |
| Cell type within a tissue | Use the named cells, rather than an entire tissue, as the reference: kidney tubule cells are High, while skeletal muscle myocytes are Not detected (HPA tissue IHC). Low staining is reported in oral squamous epithelial cells and adipocytes, so faint signal there has a different reference level (HPA tissue IHC: Low). |
| Strength of antibody evidence | Three listed antibodies have Approved IHC status (HPA antibodies: HPA028185, HPA062105, CAB004205). The tissue profile has medium agreement with RNA data and awaits external verification (HPA tissue IHC: reliability description); Approved status should therefore inform, not replace, slide-level controls (standard IHC practice). |
| Resolution of the readout | HPA’s ICC-IF summary places BAD mainly at mitochondria (HPA subcellular ICC-IF: Mitochondria, enhanced), while tissue IHC reports ubiquitous cytoplasmic expression (HPA tissue IHC: profile). Chromogenic paraffin IHC may show cytoplasmic color without resolving individual mitochondria (standard IHC interpretation). |
| Retrieval and detection workflow | Retrieval conditions, blocking, and detection can affect chromogenic IHC signal generally (standard IHC practice). No BAD-specific fixation sensitivity or retrieval requirement is established by the supplied UniProt and HPA records; do not infer one from topology, modifications, or tissue staining levels (UniProt Q92934; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control cytoplasm is blank. | The assay may have failed, or the chosen control may lack the relevant cells (standard IHC practice; HPA tissue IHC: cell-specific calls). | Confirm that kidney tubule or bronchial respiratory epithelial cells are present, then check retrieval, primary antibody, and detection controls (HPA tissue IHC: High; standard IHC practice). |
| The slide looks uniformly brown. | Nonspecific primary binding or detection background can obscure cellular staining (standard IHC practice). | Review a no-primary control, blocking, and antibody concentration; score only a defined cellular pattern (standard IHC practice). |
| Nuclei dominate the staining. | That distribution conflicts with BAD’s reported cytoplasmic and mitochondrial locations (UniProt Q92934 localization). | Compare with a positive tissue control and an independently validated antibody before assigning the nuclear signal to BAD (HPA antibodies: IHC Approved; standard IHC practice). |
| Myocytes or ovarian stroma stain strongly. | Those cells are Not detected in the HPA tissue reference, making cross-reactivity or endogenous activity plausible (HPA tissue IHC: Not detected; standard IHC practice). | Check a no-primary control and repeat with an independently validated antibody; interpret the discrepancy cautiously because HPA verification is pending (standard IHC practice; HPA tissue IHC: reliability description). |
| Expected cytoplasmic signal is faint. | Low reference expression can explain faint staining in adipocytes or oral squamous epithelial cells (HPA tissue IHC: Low); weak assay performance remains possible (standard IHC practice). | Compare the same run with a High reference cell type before changing the interpretation or general IHC conditions (HPA tissue IHC: High; standard IHC practice). |
| IF/ICC shows mitochondrial signal, but paraffin IHC looks broadly cytoplasmic. Is that inconsistent? | HPA reports mainly mitochondrial ICC-IF localization and ubiquitous cytoplasmic tissue IHC; chromogenic IHC has less spatial detail (HPA subcellular ICC-IF; HPA tissue IHC; standard IHC interpretation). | Interpret each readout against its own reference pattern; consult the separate IF/ICC guide for that application (HPA subcellular ICC-IF; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing BAD staining
M03520 · IHCIHC analysis of BAD using anti-BAD antibody (M03520). BAD was detected in a paraffin-embedded section of human breast cancer tissue. Heat me
M03520 · IHCIHC analysis of BAD using anti-BAD antibody (M03520). BAD was detected in a paraffin-embedded section of human placenta tissue. Heat mediate
M03520 · IFImmunofluorescent analysis of Hela cells, using Bad Antibody .
M03520-1 · IHCRat kidney was stained with anti-Bad rabbit antibody
M03520-1 · IHCHuman kidney was stained with anti-Bad rabbit antibody
A03520 · IHCImmunohistochemical staining of rat thymus using Bad at 2 μg/mL.
A03520 · IFImmunofluorescence of BAD in Rat Thymus cells with BAD antibody at 10 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000002330-BAD/tissue/cerebral+cortex#imid_1393436
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000002330-BAD/tissue/colon#imid_7310006
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000002330-BAD/tissue/liver#imid_7310007
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000002330-BAD/tissue/kidney#imid_7310000
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028185; green = BAD, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028185; green = BAD, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BAD IHC & IF
🧪Run kidney first: tubular cells should stain strongly (HPA: High in cells in tubules). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in myocytes); on the kidney slide, cells outside the tubules should show little or no staining relative to tubules, but their negative status requires validation (HPA reports the tubular signal only).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Ovary (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show BAD in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and species- and clonality-matched rabbit IgG isotype controls, plus BAD-knockout tissue if available (selected IHC caption: rabbit primary; standard IHC practice). For DAB detection, quench endogenous peroxidase and check kidney background with the no-primary control; check endogenous biotin if using biotin-based detection (selected IHC caption: DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M03520 paraffin-section caption does not state the fixative (selected IHC caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; compare retrieved and unretrieved sections if optimizing (selected IHC caption: EDTA retrieval; standard IHC practice). HPA has ICC-IF images showing mainly mitochondrial localization, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC (HPA subcellular: mitochondria; selected IHC caption: paraffin section).
HPA tissue IHC evidence for BAD
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
Section 3
Advanced BAD IHC Tips
Troubleshoot BAD staining in paraffin sections by checking retrieval, compartment, controls and cell-specific scoring before interpreting chromogenic signal.
What retrieval should I use when BAD staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M03520). The demonstrated stain used a 1:50 rabbit antibody incubation overnight at 4°C, so keep those conditions consistent while assessing retrieval (datasheet M03520). Compare a known positive section processed alongside the test section, and inspect both signal and tissue integrity after heating (HPA: high in kidney tubules; standard IHC practice). If staining remains weak, test a gentler or longer heating cycle on adjacent sections, changing one variable at a time (standard IHC practice). Avoid calling an absent signal biological until the positive control stains appropriately (standard IHC practice).
Could fixation explain inconsistent BAD staining across paraffin blocks?
Target-specific fixation sensitivity is unknown because the selected tissue caption identifies a paraffin-embedded section but does not state its fixative (datasheet M03520). Record the fixative, fixation duration and processing history for each block, then compare sections with matched processing where possible (standard IHC practice). Use the demonstrated EDTA retrieval at pH 8.0 and 1:50 antibody incubation overnight at 4°C as a consistent starting point (datasheet M03520). If blocks differ in signal, assess morphology and an external positive control before changing retrieval or antibody concentration (standard IHC practice). Differences between blocks alone cannot establish that BAD has a particular fixation sensitivity (standard IHC practice).
Should BAD appear diffuse or mitochondrial in chromogenic IHC?
Expect cytoplasmic staining to be plausible in tissue sections, while keeping mitochondrial association in mind (HPA: ubiquitous cytoplasmic tissue expression; UniProt Q92934: mitochondrion outer membrane and cytoplasm). HPA cell imaging reports mitochondria as the main location, but chromogenic IHC may not resolve individual mitochondria (HPA: enhanced mitochondrial location; standard IHC practice). Assess whether staining lies within the expected cells rather than scoring a fine granular pattern alone as proof of organelle localisation (standard IHC practice). Phosphorylation can shift BAD toward the cytoplasm, so compartment changes may have biological context without establishing phosphorylation from this stain (UniProt Q92934). Compare adjacent sections and a no-primary control before interpreting unusual nuclear or extracellular signal (standard IHC practice).
Can this stain distinguish BAD isoforms or phosphorylation states?
Do not assign an isoform from chromogenic intensity: the supplied record lists 0 isoforms and one annotated 1–168 chain (UniProt Q92934). BAD has no annotated transmembrane segment, but the selected antibody caption does not identify its epitope (UniProt Q92934 topology; datasheet M03520). Phosphoserines at 25, 75 and 91 make modification-dependent recognition a question to test, not a property to assume for this antibody (UniProt Q92934 modified residues; standard IHC practice). Compare staining after the same EDTA pH 8.0 retrieval, and seek epitope or phosphospecific validation before making state-specific claims (datasheet M03520; standard IHC practice). Score the result as BAD immunoreactivity unless that additional validation is available (standard IHC practice).
How can IF help check the cell and compartment assignment from BAD IHC?
Use IF as a separate corroboration experiment for the IHC cell assignment, with a marker for the expected cell type, such as respiratory epithelial cells in bronchus (HPA: high in bronchus respiratory epithelial cells; standard IF practice). Add a mitochondrial marker when testing the reported main subcellular location, while allowing cytoplasmic BAD signal (HPA: enhanced mitochondrial location; UniProt Q92934: cytoplasm). Choose spectrally separated fluorophores and assess unstained tissue autofluorescence before assigning faint signal (standard IF practice). BAD lacks an annotated transmembrane segment, but its antibody epitope and membrane-facing side are unspecified; optimise permeabilisation for access to that epitope rather than presuming an orientation (UniProt Q92934 topology; datasheet M03520; standard IF practice). Keep IF controls and conclusions distinct from the demonstrated paraffin-section chromogenic workflow (datasheet M03520; standard IF practice).
How do I separate BAD signal from diffuse brown background?
Run a no-primary control beside the test section to reveal detection-system and tissue background (standard IHC practice). The demonstrated paraffin-section stain used 10% goat serum block, a 1:50 rabbit primary overnight at 4°C, and HRP with DAB (datasheet M03520). Include a peroxidase-blocking step and check whether brown deposits persist without primary antibody before attributing them to BAD (standard chromogenic IHC practice). If background rises with primary antibody, compare a dilution series while preserving the same retrieval and exposure conditions (standard IHC practice). Judge useful signal within cells against the no-primary section and a positive tissue control, particularly when staining is widespread (HPA: ubiquitous cytoplasmic expression; standard IHC practice).
What is a defensible way to quantify BAD in chromogenic sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA describes widespread cytoplasmic tissue staining (HPA: ubiquitous cytoplasmic expression; standard IHC practice). For a cell-based readout, report the percentage of positive viable cells and an H-score using intensity grades 0–3, yielding 0–300 (standard IHC practice). If counting discrete positive cells, report density per mm² of viable tissue, with the region and area stated (standard IHC practice). Normalise comparisons to the same eligible cell type or viable tissue area, and use identical retrieval, detection and scoring thresholds across sections (standard IHC practice). Record cytoplasmic staining separately from ambiguous edge, necrotic or extracellular deposits rather than folding them into the score (standard IHC practice).
When should an apparent BAD-positive area be treated as artefact?
Treat cytoplasmic staining in the expected cells as more credible than isolated nuclear or extracellular deposits (HPA: ubiquitous cytoplasmic expression; UniProt Q92934: cytoplasm and mitochondrial outer membrane; standard IHC practice). Check cell identity against the section, since HPA reports high staining in kidney tubules but no detection in ovarian stroma cells (HPA: kidney tubules high; ovarian stroma cells not detected). Exclude section-edge intensification, necrotic regions and deposits reproduced in the no-primary control from biological interpretation (standard IHC practice). Brown signal caused by endogenous peroxidase should diminish after an appropriate peroxidase block (standard chromogenic IHC practice). Interpret a clean positive cautiously because HPA rates its tissue stain approved with medium RNA consistency and pending external verification (HPA: reliability description).
Boster reagents
Best BAD / Bcl2-associated agonist of cell death IHC Antibodies
These anti-BAD antibodies list human, mouse and rat reactivity (catalog: reactivity). IHC images cover human breast cancer, kidney and rat thymus; IF images cover HeLa and rat thymus cells (catalog: image captions).
Real IHC data
Anti-Bad Rabbit Monoclonal Antibody
Cat # M03520
Real IHC data
Anti-Bad Rabbit Monoclonal Antibody
Cat # M03520-1
Real IHC data
Anti-BAD Antibody
Cat # A03520
M03520 has IHC images of human breast cancer, human placenta and mouse small intestine, plus an IF image of HeLa cells (M03520: image captions); M03520-1 has IHC images of human, mouse and rat kidney (M03520-1: image captions). A03520 has IHC and IF images of rat thymus samples (A03520: image captions).
Which to pick: For paraffin-section IHC, start with M03520 at 1:50 after EDTA retrieval at pH 8.0 (M03520: IHC caption); the caption does not report the fixative (M03520: IHC caption). For IF/ICC, choose M03520 because ICC and IF are listed applications and an IF image shows HeLa cells (M03520: applications and IF caption). For cross-species tissue IHC, choose M03520-1: its IHC images show human, mouse and rat kidney, with a listed dilution of 1:200–1:1000 (M03520-1: IHC captions and catalog dilution).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q92934 (BAD_HUMAN, Bcl2-associated agonist of cell death).
- Human Protein Atlas. BAD tissue IHC expression (reliability: Approved).
- Human Protein Atlas. BAD subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the principal piece and end piece..
- Human Protein Atlas. BAD antibody validation summary (3 antibodies).
- Expression and prognostic relevance of MET and phospho-BAD in non-small cell lung cancer. OncoTargets and therapy 2013 — PMC3787924.
- Role of FoxO1 and apoptosis in pulmonary vascular remolding in a rat model of chronic thromboembolic pulmonary hypertension. Scientific reports 2017 — PMC5442111.
- Digital spatial profiling identifies the tumor center as a topological niche in prostate cancer characterized by an upregulation of BAD. Scientific reports 2024 — PMC11366015.
- Astaxanthin alleviates early brain injury following subarachnoid hemorrhage in rats: possible involvement of Akt/bad signaling. Marine drugs 2014 — PMC4145317.
- PubMed PMID:8929532 — UniProt-cited evidence.
- PubMed PMID:9388232 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BAD (Bcl2-associated agonist of cell death). Expected localisation Cytoplasm (HPA tissue IHC); mitochondrial outer membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03520). Validated IHC antibody: M03520 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Ovary. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet M03520); 2. Fixation: Image fixative and duration unreported (datasheet M03520); 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 M03520); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03520); 8. Primary antibody: Rabbit monoclonal (clone CHC-2) anti-BAD, 1:50 (datasheet M03520); 9. Primary incubation: Overnight at 4 °C (datasheet M03520). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03520); Caveat: Phosphorylation can shift BAD toward the cytoplasm (UniProt); Isoform / epitope: No annotated isoforms; one full-length chain (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt Q92934, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet M03520, PMC3787924, PMC5442111. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BAD/immunohistochemistry-ihc-guide.