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APAF1 / Apoptotic protease-activating factor 1 · IHC design guide
Design Immunohistochemistry for APAF1
Plan APAF1 staining in paraffin sections using its general cytoplasmic tissue pattern and strong signal in bone marrow hematopoietic cells (HPA tissue IHC). This guide covers fixation consistency, antibody selection and interpretation across cell populations.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
APAF1 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 | General cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal, strongest in subsets of immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Cell mix can skew signal toward hematopoietic cells (HPA tissue IHC) | |
| Regulation | Tissue-dependent expression (HPA tissue IHC) | |
| Isoform / epitope | 6 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended APAF1 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol (datasheet) with a published APAF1 protocol for FFPE solitary fibrous tumor sections (PMC6746689 methods).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00889); 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 anti-APAF1, 1 μg/mL (datasheet A00889) |
| 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 | APAF1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression, most abundant in subsets of immune cells. 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; UniProt O14727: cytoplasm). The paper also used pH 9.0 buffer (PMC6746689 methods).
Section 2
What Is the Expected APAF1 Staining Pattern?
APAF1 is a cytoplasmic protein without a transmembrane segment (UniProt O14727: subcellular location and topology). In paraffin-section IHC, expect cytoplasmic staining across several cell types, strongest in subsets of immune cells; HPA reports High staining in bone marrow hematopoietic cells and Medium staining in selected glandular and endothelial cells (HPA: tissue IHC). HPA rates the tissue profile Enhanced, while noting medium consistency with RNA data and staining in some unannotated structures (HPA: reliability description).
| Cytoplasmic signal is strongest in bone marrow hematopoietic cells, with weaker signal in other cells. | This fits the reported general cytoplasmic pattern and High hematopoietic staining (HPA: tissue IHC). Compare cells within the section: HPA reports Medium staining in appendix and duodenum glandular cells and colon endothelial cells, so equally intense staining throughout every compartment warrants review (HPA: tissue IHC). |
| Predominantly nuclear or membrane outlining staining appears in paraffin sections. | Recheck compartment scoring and controls: cytoplasm is the UniProt location and the general HPA tissue IHC pattern (UniProt O14727: subcellular location; HPA: tissue IHC). HPA ICC-IF also reports nucleoplasm and Golgi localization, so nuclear signal alone cannot prove artefact across methods (HPA: subcellular ICC-IF). |
| Strong signal appears in a cell type HPA lists as undetected. | For example, HPA reports APAF1 as not detected in adipocytes and cardiomyocytes; these are cell-specific observations, not negative calls for entire tissues (HPA: tissue IHC). Check whether staining follows cell boundaries and survives appropriate detection controls; cross-reactivity or endogenous detection activity are possible explanations, not diagnoses from appearance alone (general IHC practice). |
| Diffuse staining obscures cell boundaries or fills the section uniformly. | A uniform haze cannot establish APAF1-positive cells or cytoplasmic localization (general IHC interpretation). Assess a no-primary control and inspect blocking, antibody concentration, washes and chromogen development for nonspecific signal (general chromogenic IHC practice). Preserve the cell-type comparison because the reported pattern varies within tissues (HPA: tissue IHC). |
| Bone marrow hematopoietic cells show no discernible signal. | That conflicts with HPA’s High staining in those cells, but does not by itself show APAF1 is absent from the specimen (HPA: tissue IHC). Verify that hematopoietic cells are present and that the detection run worked, then assess retrieval and antibody conditions using the catalog antibody’s IHC-P instructions (general IHC practice). |
💡Expected APAF1 appearanceCall positive IHC when cytoplasmic staining is clear in bone marrow hematopoietic cells at the reported High level, allowing Medium staining in documented glandular or endothelial cells; uniform haze or staining confined to an unexpected compartment needs control review (HPA: tissue IHC; UniProt O14727: cytoplasm; general IHC interpretation).
| Cell type and tissue choice | Bone marrow hematopoietic cells offer the clearest reported positive comparison (High); appendix and duodenum glandular cells and colon endothelial cells are Medium (HPA: tissue IHC). Use the named cell populations when interpreting a section, since HPA’s undetected calls also refer to specific cells rather than whole organs (HPA: tissue IHC). |
| Subcellular evidence differs by application | UniProt assigns cytoplasm, and HPA describes general cytoplasmic tissue IHC (UniProt O14727: subcellular location; HPA: tissue IHC). HPA ICC-IF additionally reports nucleoplasm and Golgi localization; that observation should be read as ICC-IF evidence, not assumed to define the chromogenic paraffin-section pattern (HPA: subcellular ICC-IF). |
| Antibody validation scope | HPA lists HPA031373 as IHC Enhanced and CAB069399 as IHC Approved; HPA031373 has ICC Uncertain (HPA: antibodies). Those labels support different levels of application-specific confidence and do not establish that an arbitrary catalog antibody reproduces either pattern (HPA: antibodies; general assay interpretation). Match the antibody and application before using an image as a comparator. |
| Isoform coverage | UniProt lists 6 APAF1 isoforms, but the supplied record does not map the catalog antibody’s epitope to them (UniProt O14727: isoforms). A staining difference cannot be assigned to a particular isoform from these data; check antibody epitope information if isoform coverage matters (general antibody interpretation). |
| Protein topology and state | APAF1 has no transmembrane segment and forms an apoptosome after cytochrome c and dATP binding (UniProt O14727: topology and subunit). Interpret a membrane-rim pattern cautiously; routine chromogenic staining also cannot distinguish monomeric APAF1 from assembled apoptosome on morphology alone (UniProt O14727: topology and subunit; general IHC interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the expected positive cells | The positive cell population may be absent from the examined field, or the IHC detection run may have failed (general IHC practice). HPA reports High staining specifically in bone marrow hematopoietic cells (HPA: tissue IHC). | Confirm cell identity on the section, review a working positive control, then follow the catalog antibody’s IHC-P instructions while checking retrieval, primary incubation and detection (general IHC practice). Do not infer APAF1 absence from a failed control. |
| Strong staining in HPA-undetected cells | Cross-reactivity, endogenous enzyme activity or nonspecific detection may contribute (general chromogenic IHC practice). HPA’s undetected entries are cell-specific, such as adipocytes or cardiomyocytes (HPA: tissue IHC). | Identify the stained cell type, inspect a no-primary control and the antibody’s validated IHC application, and compare with a reported positive population (general IHC practice; HPA: tissue IHC). Do not score the entire organ as negative based on one cell-type entry. |
| Diffuse brown background limits scoring | Nonspecific binding, insufficient washing or excess chromogen development can obscure cellular contrast (general chromogenic IHC practice). | Compare the no-primary control, review blocking and washes, and adjust antibody concentration or development within the validated IHC-P workflow (general IHC practice). Score APAF1 only when cellular cytoplasm can be separated from background (UniProt O14727: cytoplasm; general IHC interpretation). |
| Signal appears mainly nuclear in IHC | Compartment assignment or detection artefact needs review against the reported cytoplasmic tissue pattern (HPA: tissue IHC). Nuclear localization is nevertheless reported in ICC-IF (HPA: subcellular ICC-IF). | Check the counterstain and nuclear boundaries, compare a control section, and report nuclear IHC signal separately from cytoplasmic scoring (general IHC practice). Avoid transferring the ICC-IF location call directly to paraffin-section IHC (HPA: tissue IHC; HPA: subcellular ICC-IF). |
| Two antibodies give different IHC patterns | Application validation differs: HPA031373 is IHC Enhanced and CAB069399 IHC Approved (HPA: antibodies). Their labels do not establish identical epitope recognition (HPA: antibodies; general antibody interpretation). | Compare each antibody’s documented IHC performance in the same reported positive cell population, then examine controls and epitope information where available (HPA: tissue IHC; general IHC practice). Record which antibody produced each compartment and cell-type pattern. |
| Can the ICC-IF pattern set the IHC scoring rule? | HPA ICC-IF reports nucleoplasm and cytosol as main locations, plus Golgi, whereas tissue IHC is described as generally cytoplasmic (HPA: subcellular ICC-IF; HPA: tissue IHC). | Use the tissue IHC pattern to score this paraffin-section assay; consult the separate IF/ICC guide for fluorescence design (HPA: tissue IHC; HPA: subcellular ICC-IF). HPA031373 is IHC Enhanced but ICC Uncertain, so its IHC status should not be read as ICC validation (HPA: antibodies). |
Experiment evidence
IHC & IF images showing APAF1 staining
A00889 · IHCImmunohistochemistry of Apaf1 in human heart tissue with Apaf1 antibody at 1 μg/mL.
A00889 · IFImmunofluorescence of Apaf1 in K562 cells with Apaf1 antibody at 10 μg/mL.
M00889 · IHCImmunohistochemical analysis of paraffin-embedded mouse kidney, using APAF1 Antibody .
M00889-2 · IHCImmunohistochemistry of Apaf1 in K562 cells with Apaf1 antibody at 0.5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000120868-APAF1/tissue/cerebral+cortex#imid_18526403
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000120868-APAF1/tissue/colon#imid_18526345
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000120868-APAF1/tissue/liver#imid_18526330
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000120868-APAF1/tissue/kidney#imid_18526339
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA031373; green = APAF1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA031373; green = APAF1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for APAF1 IHC & IF
🧪Run bone marrow first and expect hematopoietic cells to stain (High; HPA: bone marrow). Use adipose tissue as the negative comparison, where adipocytes are Not detected (HPA: adipose tissue); no verified internal negative cell type is identified for bone marrow, so any cells without signal should be treated as background comparators rather than confirmed APAF1-negative cells (HPA: bone marrow hematopoietic cells High).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show APAF1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host and clonality, and APAF1 knockout material or cognate peptide blocking to test target-dependent staining (standard IHC practice). In bone marrow, quench endogenous peroxidase for chromogenic detection and check endogenous biotin background if using avidin–biotin detection (standard IHC practice; HPA: bone marrow hematopoietic cells High).
⚠️Feasibility: No APAF1-specific fixation window or fixation effect is reported in the supplied evidence, and the A00889 heart-tissue IHC caption does not state a fixative (selected A00889 IHC caption). Antigen-retrieval dependence and whether frozen sections or IF are easier are unreported; the available ICC-IF images do not provide a matched processing comparison (HPA subcellular). Endogenous peroxidase in bone marrow hematopoietic cells can complicate chromogenic interpretation, so assess staining against the no-primary control (standard IHC practice; HPA: bone marrow hematopoietic cells High).
HPA tissue IHC evidence for APAF1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced APAF1 IHC Tips
Troubleshoot APAF1 staining in paraffin sections by checking retrieval, cell compartment, tissue context and controls before interpreting chromogenic signal.
Which retrieval conditions should I start with for APAF1 in paraffin sections?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Keep section thickness, cooling and antibody incubation consistent while comparing retrieval runs, so changes in staining can be assigned to retrieval (standard IHC practice). Because APAF1 is cytoplasmic and has no transmembrane segment, assess diffuse cellular staining against tissue morphology rather than expecting a membrane rim (UniProt O14727). If signal is weak, compare a modestly longer citrate retrieval with the starting condition, watching for tissue damage and increased background (standard IHC practice). Include a known positive tissue region and a no-primary control in that comparison (standard IHC practice).
How should I troubleshoot weak APAF1 staining when fixation history varies?
Record the fixative, fixation duration and processing history for each paraffin block before comparing APAF1 intensity (standard IHC practice). Target-specific fixation sensitivity is unknown from the supplied evidence; the selected heart-tissue image reports 1 µg/mL antibody but does not state its fixative (caption: A00889). Compare differently processed sections using the same citrate pH 6.0 retrieval and detection run, with a positive tissue control on each slide (page retrieval rule; standard IHC practice). If weak signal follows one processing batch, check section adhesion, morphology and control staining before attributing the difference to APAF1 biology (standard IHC practice). Avoid inferring fixation tolerance from tissue expression patterns or protein topology (standard IHC practice).
What cellular staining pattern is credible for APAF1?
Expect cytoplasmic APAF1 staining as the primary pattern in chromogenic sections (UniProt O14727: cytoplasm; HPA tissue IHC: general cytoplasmic expression). HPA cell imaging also reports nucleoplasm and cytosol as main locations, with additional Golgi localisation, so a nuclear component warrants validation rather than automatic rejection (HPA subcellular). Compare staining in intact cells with nuclear counterstain and tissue architecture; isolated pigment, extracellular deposits or a uniform slide-wide haze do not establish cellular localisation (standard IHC practice). APAF1 has no transmembrane segment, so a crisp membrane-only outline is discordant with its recorded topology (UniProt O14727). Recheck that pattern with a no-primary control and an independently validated antibody when available (standard IHC practice).
Could epitope choice explain different APAF1 staining patterns?
Check the catalog antibody's documented immunogen or epitope before comparing staining across experiments, because APAF1 has 6 recorded isoforms (UniProt O14727). The supplied record identifies a CARD domain at residues 1–90 and an NB-ARC domain at 104–415, but gives no antibody epitope (UniProt O14727; supplied antibody evidence). Therefore, the observed chromogenic pattern cannot establish which isoform is present or whether an epitope is shared across isoforms (standard IHC interpretation). Compare serial sections with an independently validated antibody against a documented different region, if available, while keeping retrieval and scoring consistent (standard IHC practice). Treat differences as candidates for follow-up, not proof of isoform-specific expression (standard IHC interpretation).
How can IF help resolve ambiguous APAF1 IHC localisation?
On the separate IF/ICC workflow, multiplex APAF1 with a validated hematopoietic-cell marker when examining marrow, where HPA reports high staining in hematopoietic cells (HPA tissue IHC). Choose spectrally separated fluorophores and place the weaker signal in a channel with lower tissue autofluorescence; inspect unstained and single-stain controls (standard IF practice). Permeabilise sufficiently to reach a cytosolic epitope, since APAF1 is cytoplasmic and lacks a transmembrane segment, while confirming the antibody's epitope accessibility experimentally (UniProt O14727; standard IF practice). Compare cytosolic signal with the nuclear counterstain because HPA cell images also report nucleoplasmic localisation (HPA subcellular). Use IF as a localisation check alongside, rather than a numerical substitute for, chromogenic tissue scoring (standard IHC/IF practice).
How do I distinguish APAF1 signal from chromogenic background?
Run a no-primary control and check that peroxidase blocking suppresses endogenous enzyme signal before reading DAB staining (standard chromogenic IHC practice). Compare the control with intact-cell staining in the expected cytoplasmic compartment, especially in immune-cell-rich regions where HPA describes abundant APAF1 (UniProt O14727; HPA tissue IHC). If diffuse haze rises with the antibody concentration, optimise dilution and washing while holding retrieval at citrate pH 6.0 initially (page retrieval rule; standard IHC practice). Examine edges, folds, necrotic areas and pigment separately because they can distort apparent positive area (standard IHC practice). Record whether the background occurs across all sections or only one processing batch before changing antibody conditions (standard IHC practice).
How should I score APAF1 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell compartment and eligible cell population before scoring, then apply the same rule to every section (standard IHC practice). For cellular staining, report the percentage of positive eligible cells and an H-score using intensity categories 0–3 multiplied by their percentages, giving 0–300 (standard IHC scoring). For sparse positive populations, report positive-cell density per mm² of viable tissue or a defined compartment, alongside its denominator (standard histologic quantification). Normalise comparisons to eligible cells or viable tissue area, with matched section processing, imaging and thresholds (standard IHC practice). Because HPA reports high marrow hematopoietic-cell staining but variable staining elsewhere, retain cell-type annotations instead of pooling all cells into one score (HPA tissue IHC).
When does APAF1 staining support a biological conclusion?
Call staining credible when it maps to intact cells, follows a plausible cytoplasmic distribution and exceeds the matched no-primary control (UniProt O14727; standard IHC practice). High signal in marrow hematopoietic cells fits the HPA tissue profile, whereas cardiomyocytes are listed as not detected despite a selected heart-tissue staining image; identify the stained cell population before reconciling these observations (HPA tissue IHC; caption: A00889). Reject edge effects, necrotic deposits and residual endogenous peroxidase as evidence of APAF1 expression when controls reveal them (standard IHC practice). APAF1 can assemble into the apoptosome, but chromogenic abundance alone cannot show oligomerisation, caspase activation or apoptosis (UniProt O14727; standard IHC interpretation). Interpret intensity with morphology and independent functional evidence when making those claims (standard IHC practice).
Boster reagents
Best APAF1 / Apoptotic protease-activating factor 1 IHC Antibodies
APAF1 IHC images show human heart tissue, paraffin-embedded mouse kidney, and K562 cells; an IF image shows K562 cells (catalog image captions). Listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-Apaf1 Antibody
Cat # A00889
Real IHC data
Anti-APAF1 Rabbit Monoclonal Antibody
Cat # M00889
Real IHC data
Anti-Apaf-1 Monoclonal Antibody [2E10]
Cat # M00889-2
A00889 has a human-heart IHC image and a K562-cell IF image; its listed reactivity is human, mouse, and rat (catalog image captions; applications; reactivity). M00889 has a paraffin-embedded mouse-kidney IHC image and lists IF/ICC, while M00889-2 has a K562-cell IHC image and lists IHC-P (catalog image captions; applications).
Which to pick: For tissue IHC, choose A00889 for the human-heart example or M00889 for the paraffin-embedded mouse-kidney example; the M00889 caption does not report a fixative (catalog IHC image captions). For IF/ICC, choose M00889 when ICC is required (catalog applications), or A00889 when a matching IF image is useful (catalog K562-cell IF image caption). For human, mouse, and rat reactivity, choose rabbit A00889 if IF is also needed, or mouse monoclonal M00889-2 for IHC-P; its IHC image shows K562 cells (catalog reactivity; host; clone; applications; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O14727 (APAF_HUMAN, Apoptotic protease-activating factor 1).
- Human Protein Atlas. APAF1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. APAF1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to the Golgi apparatus..
- Human Protein Atlas. APAF1 antibody validation summary (2 antibodies).
- Molecular changes in solitary fibrous tumor progression. Journal of molecular medicine (Berlin, Germany) 2019 — PMC6746689.
- Apoptosome-dependent caspase activation proteins as prognostic markers in Stage II and III colorectal cancer. British journal of cancer 2012 — PMC3341858.
- Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma. Nature medicine 2015 — PMC4775273.
- Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function. Frontiers in immunology 2019 — PMC6892980.
- PubMed PMID:9267021 — UniProt-cited evidence.
- PubMed PMID:10441496 — UniProt-cited evidence.
- PubMed PMID:10364241 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for APAF1 (Apoptotic protease-activating factor 1). Expected localisation General cytoplasmic staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00889 at 1 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A00889); 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 anti-APAF1, 1 μg/mL (datasheet A00889); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Cell mix can skew signal toward hematopoietic cells (HPA tissue IHC); Isoform / epitope: 6 isoforms; check antibody epitope coverage (UniProt); Regulation: Tissue-dependent expression (HPA tissue IHC). Evidence: UniProt O14727, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A00889, PMC6746689. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/APAF1/immunohistochemistry-ihc-guide.