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ALK / ALK tyrosine kinase receptor · IHC design guide
Design Immunohistochemistry for ALK
Plan ALK chromogenic IHC in paraffin sections using cerebellar Purkinje cells, which show high staining, as a positive reference (HPA tissue IHC). Compare the expected membrane location (UniProt) with observed cytoplasmic and nuclear tissue staining, and assess possible off-target binding (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ALK 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 | Membrane expected (UniProt); cytoplasm and nuclei observed (HPA tissue IHC) | |
| Staining pattern | Neurons show cytoplasmic and nuclear staining (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 | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target binding may mimic ALK staining (HPA tissue IHC) | |
| Regulation | Transient in select neural regions (UniProt) | |
| Isoform / epitope | No isoforms listed; extracellular versus cytoplasmic epitope matters (UniProt) |
Section 1
Recommended ALK IHC & IF Protocols
The catalog antibody protocol is accompanied by one published ALK IHC method using FFPE tissue sections (PMC5890087).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet M00301-5); 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-ALK, 1:200-1:1000 (datasheet M00301-5) |
| 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 | ALK-positive staining in purkinje cells of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in several different tissue types, most abundant in neurons. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); the published method uses T/E buffer (PMC5890087).
Section 2
What Is the Expected ALK Staining Pattern?
ALK is a single-pass cell-membrane receptor with an extracellular region and a cytoplasmic kinase region (UniProt Q9UM73 topology). In IHC, the strongest reported staining is in cerebellar Purkinje cells and neurons of the cerebral cortex and hippocampus (HPA: High). HPA also reports cytoplasmic and nuclear tissue staining, but rates tissue IHC Approved with low agreement between staining and RNA data and notes presumed off-target binding (HPA: tissue IHC reliability).
| Distinct staining in Purkinje cells or cortical and hippocampal neurons, with staining at the cell edge and possibly within the cell (HPA: High in these cells; HPA: tissue profile). | This is the most persuasive tissue pattern for ALK IHC. Membrane staining fits receptor topology, while cytoplasmic staining has also been reported in tissue sections (UniProt Q9UM73 topology; HPA: tissue profile). Interpret intensity alongside cell identity and the negative control; HPA's Approved rating does not resolve its reported staining–RNA disagreement (HPA: tissue IHC reliability; general IHC practice). |
| Predominantly nuclear staining, especially without a discernible neuronal or membrane-associated pattern (HPA: tissue profile; UniProt Q9UM73 subcellular location). | Do not treat nuclear signal alone as proof of correctly localized ALK. HPA reports nuclear staining in tissue IHC, whereas UniProt assigns ALK to the cell membrane and HPA ICC-IF supports plasma-membrane localization (HPA: tissue profile; UniProt Q9UM73 subcellular location; HPA: subcellular). Review the full cell pattern and controls before assigning specificity (general IHC practice). |
| Strong staining in an unexpected cell population, such as adipocytes or pancreatic exocrine glandular cells (HPA: Not detected in those cells). | Compare the exact cell type rather than labeling the whole organ positive or negative: HPA reports no detection in these specified populations and presumed off-target tissue binding (HPA: tissue IHC; HPA: tissue IHC reliability). Cross-reactivity or endogenous chromogen-generating activity should be considered if the signal also appears in appropriate controls (general IHC practice). |
| Diffuse color across many cell types, tissue spaces, or the section background, with little separation between neurons and surrounding tissue (general IHC practice). | That distribution provides weak evidence for cell-specific ALK staining. Check whether the same color appears in a no-primary control, then assess blocking, washing, detection reagents, and antibody concentration as general chromogenic IHC variables (general IHC practice). HPA's reported off-target binding adds a reason to require convincing cellular contrast (HPA: tissue IHC reliability). |
| No visible staining in Purkinje cells or cortical or hippocampal neurons on a section expected to contain them (HPA: High in these cells). | First verify that the relevant cells are present and preserved, then check the antibody's documented IHC-P procedure, detection reagents, and a concurrently processed positive control (general IHC practice). Absence of signal can reflect assay failure or sample variation; HPA's High observations do not establish that every section must stain identically (HPA: tissue IHC). |
💡Expected ALK appearanceCall a result supportive when High staining preferentially marks identifiable Purkinje cells or cortical or hippocampal neurons, ideally with a membrane-associated component; broad background or staining confined to unexpected cells weakens that call (HPA: tissue IHC and tissue profile; UniProt Q9UM73 topology; general IHC practice).
| Cell and tissue selection (HPA: tissue IHC; UniProt Q9UM73 tissue specificity). | Cerebellar Purkinje cells and cerebral cortical and hippocampal neurons are reported High, while caudate neurons and skin keratinocytes are Medium (HPA: tissue IHC). UniProt reports expression in brain and CNS, small intestine, and testis, and absence from normal lymphoid cells; these are expression statements, not guaranteed IHC results in every specimen (UniProt Q9UM73 tissue specificity). |
| Antibody validation and interpretation (HPA: HPA010694 validation; HPA: tissue IHC reliability). | HPA010694 is Approved for IHC and Supported for ICC, but HPA describes low consistency between tissue staining and RNA expression and disregarded presumed off-target binding (HPA: antibody validation; HPA: tissue IHC reliability). Treat a positive tissue stain as evidence to assess with morphology and controls, not as independent confirmation of target identity (general IHC practice). |
| Compartment and epitope position (UniProt Q9UM73 topology; HPA: subcellular). | Full-length ALK has extracellular residues 19–1038, a transmembrane segment at 1039–1059, and cytoplasmic residues 1060–1620 (UniProt Q9UM73 topology). HPA ICC-IF supports plasma-membrane localization, while HPA tissue IHC describes cytoplasmic and nuclear staining (HPA: subcellular; HPA: tissue profile). The antibody epitope is unspecified here, so a domain-specific staining prediction cannot be made. |
| Antigen retrieval and detection settings (general IHC practice). | Use the chosen IHC-validated antibody's documented paraffin-section procedure and compare retrieval, antibody concentration, and chromogenic detection against controls when optimizing the assay (general IHC practice). Neither supplied source gives a target-specific retrieval condition or shows that fixation alters ALK epitope access or sensitivity; do not infer one from topology or HPA staining levels (UniProt Q9UM73; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in an identifiable Purkinje-cell or cortical-neuron positive control (HPA: High in those cells). | The assay may have failed, or the relevant cells or antigen may be poorly represented in that section (general IHC practice). | Confirm morphology and control labeling, then review the documented IHC-P procedure, retrieval step, primary-antibody preparation, and detection reagents (general IHC practice). Do not infer an ALK-specific fixation effect from this result. |
| Neurons stain, but the entire section is similarly brown (HPA: High in selected neurons; general IHC practice). | Poor signal separation may arise from nonspecific antibody binding, insufficient washing, or detection background (general IHC practice); HPA also reports presumed off-target tissue binding (HPA: tissue IHC reliability). | Inspect a no-primary control, wash and block according to the assay protocol, and optimize antibody concentration using neuronal and low-signal cell populations on the same run (general IHC practice; HPA: tissue IHC). |
| An unexpected population stains more strongly than the reported neuronal positives (HPA: High in Purkinje and cortical or hippocampal neurons). | Cell-type misidentification or off-target staining is possible; HPA's tissue reliability note specifically records presumed off-target binding (HPA: tissue IHC reliability; general IHC practice). | Recheck morphology and the exact HPA cell-level comparison. For example, HPA reports adipocytes and pancreatic exocrine glandular cells as Not detected (HPA: tissue IHC). Compare controls before interpreting the unexpected population as ALK positive (general IHC practice). |
| Signal is chiefly nuclear, with little cell-edge staining (HPA: tissue profile; UniProt Q9UM73 subcellular location). | HPA has observed nuclear tissue staining, but the receptor's annotated location and supported ICC-IF location are at the membrane; nuclear signal alone leaves specificity uncertain (HPA: tissue profile and subcellular; UniProt Q9UM73 subcellular location). | Assess whether the same cells show a coherent neuronal pattern, compare the no-primary control, and report nuclear staining as an observation rather than definitive ALK localization (HPA: tissue profile; general IHC practice). |
| Chromogen appears in the no-primary control (general IHC practice). | The color can come from endogenous detection activity or the detection system rather than primary-antibody recognition (general chromogenic IHC practice). | Address the control-positive step in the detection workflow, including appropriate endogenous-activity blocking where applicable, before scoring ALK staining (general chromogenic IHC practice). |
| IHC tissue staining seems inconsistent with membrane-focused IF images (HPA: tissue profile; HPA: subcellular). | The source observations come from different applications: HPA tissue IHC reports cytoplasmic and nuclear expression, while its ICC-IF summary supports plasma-membrane localization (HPA: tissue IHC; HPA: subcellular). | Interpret each assay against its own controls and documented localization. Use the separate IF/ICC guide for IF setup; an ICC-supported antibody designation does not supply an IF protocol for this paraffin IHC guide (HPA: antibody validation; general IHC practice). |
Experiment evidence
IHC & IF images showing ALK staining
M00301-5 · IHCHuman non-hodgkin lymphoma was stained with anti-ALK rabbit antibody
M00301-5 · IHCHuman neuroblastoma was stained with anti-ALK rabbit antibody
M00301-3 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using ALK Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000171094-ALK/tissue/cerebral+cortex#imid_2904800
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000171094-ALK/tissue/cerebellum#imid_2904795
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000171094-ALK/tissue/colon#imid_2903206
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000171094-ALK/tissue/liver#imid_2903207
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA010694; green = ALK, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA010694; green = ALK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ALK IHC & IF
🧪Run cerebellum first and expect staining in Purkinje cells (HPA: High in Purkinje cells). Use adipose tissue as the negative tissue, with adipocytes expected to show no detectable staining (HPA: Not detected in adipocytes); on the cerebellar slide, unstained cells outside the Purkinje cell population provide an internal background reference, but their ALK status is unreported in the supplied HPA row.
Positive control tissue: Cerebellum (Purkinje cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ALK in Rh30, SH-SY5Y, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and clonality-matched isotype controls; the captioned antibody is rabbit-derived (selected tissue-IHC caption: anti-ALK rabbit antibody). An ALK-knockout specimen can serve as a biological negative control, with loss of staining supporting specificity (standard IHC practice). For chromogenic detection, block endogenous peroxidase in the positive tissue; for IF, assess neuronal autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; optimize retrieval for the chosen paraffin-section assay (standard IHC practice). The selected M00301-5 tissue-IHC caption does not report a fixative (selected tissue-IHC caption: fixative not stated). The evidence does not establish whether frozen sections or IF would be easier; neuronal autofluorescence can complicate IF assessment in cerebellum (standard IF practice).
HPA tissue IHC evidence for ALK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Skin | Keratinocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ALK IHC Tips
Troubleshoot ALK staining in paraffin sections by checking retrieval, antibody specificity, expected cell populations and subcellular pattern before scoring chromogenic signal.
How should I retrieve ALK antigen when paraffin sections stain weakly?
Start ALK retrieval with citrate pH 6.0 HIER at 95–98 °C for 20 min (page IHC-P retrieval specification). Run a known positive section alongside the test section so a failed retrieval run can be distinguished from low expression (standard IHC practice; HPA: high staining in cerebellar Purkinje cells). If staining remains weak, compare a separately validated retrieval condition while holding the antibody and detection steps constant; do not treat an alternative buffer as established for this antibody (standard IHC practice). Check whether improvement preserves the expected cell pattern and membrane-associated localisation, since stronger widespread staining alone does not establish ALK specificity (UniProt Q9UM73 localisation; HPA subcellular: plasma membrane; standard IHC practice).
Could fixation explain weak or uneven ALK staining in my sections?
ALK-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue-IHC caption does not state its fixative (M00301-5 caption). Record the fixative, fixation interval and processing history for each section before comparing staining across specimens (standard IHC practice). Process a known positive control with the same workflow and compare matched sections using identical citrate pH 6.0 retrieval at 95–98 °C for 20 min (page IHC-P retrieval specification; standard IHC practice). If signal varies with processing history, report that association as an assay observation, without attributing a particular fixation effect to ALK from the available evidence (standard IHC practice).
Which ALK staining compartments should I accept in chromogenic IHC?
Full-length ALK is a membrane receptor with an extracellular region at residues 19–1038, a transmembrane segment at 1039–1059 and a cytoplasmic region at 1060–1620 (UniProt Q9UM73 topology). Plasma membrane localisation is supported independently by ICC/IF imaging (HPA subcellular: plasma membrane). Assess crisp membrane-associated staining in the expected cells, while documenting cytoplasmic or nuclear signal separately because the HPA tissue-IHC profile reports both and warns of presumed off-target binding (HPA tissue IHC: profile and reliability description; standard IHC practice). Use the same compartment definitions for positive controls and study sections, and avoid calling diffuse nuclear staining definitive full-length ALK without orthogonal confirmation (UniProt Q9UM73 topology; standard IHC practice).
How can epitope location affect interpretation of ALK IHC?
Map the antibody epitope before assigning a staining pattern to full-length ALK: its extracellular region spans 19–1038 and its cytoplasmic region spans 1060–1620 (UniProt Q9UM73 topology). The supplied record lists 0 isoforms and does not identify this catalog antibody’s epitope, so an isoform-specific interpretation is unsupported (UniProt Q9UM73 isoforms; supplied catalog evidence). Extracellular epitopes may be affected by the receptor’s reported glycosylation sites, whereas cytoplasmic epitopes include reported phosphotyrosine sites; neither observation establishes this antibody’s staining sensitivity (UniProt Q9UM73 modifications; standard IHC practice). Confirm the mapped immunogen and compare an independent epitope or assay before interpreting unusual staining as a distinct ALK species (standard IHC practice).
How should I investigate ALK localisation with complementary IF imaging?
Use the separate IF/ICC guide for imaging conditions; this page’s curated workflow concerns chromogenic IHC in paraffin sections (application specification). Multiplex ALK with a validated neuronal marker when examining neural samples, since HPA reports high staining in Purkinje and other neuronal cells (HPA tissue IHC: positive cells; standard IF practice). Choose spectrally separated fluorophores after checking tissue autofluorescence, and include single-stain controls to identify bleed-through (standard IF practice). Match permeabilisation to the mapped epitope: intracellular access is needed for a cytoplasmic epitope, whereas strong permeabilisation can obscure a membrane boundary when assessing an extracellular epitope (UniProt Q9UM73 topology; standard IF practice).
How do I distinguish ALK signal from chromogenic background?
Compare the ALK section with a no-primary control and inspect whether pigment or staining persists without primary antibody (standard IHC practice). Include a peroxidase block when using an enzyme-based DAB workflow, and check endogenous enzyme activity in the control; these are general detection safeguards rather than ALK-specific findings (standard IHC practice). Titrate the catalog antibody against a positive control while keeping citrate pH 6.0, 95–98 °C, 20 min retrieval constant (page IHC-P retrieval specification; standard IHC practice). Treat broad staining across unrelated compartments cautiously because HPA reports low consistency with RNA and presumed off-target binding in its tissue-IHC data (HPA tissue IHC: reliability description).
What should I measure when scoring ALK IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the eligible cell population and staining compartment before scoring, using the same criteria across all sections (standard IHC practice). Record the percentage of positive eligible cells and intensity grades, then calculate an H-score if intensity distribution matters; alternatively, report positive-cell density per mm² of viable tissue (standard IHC practice). Normalise counts to the number of eligible cells or sampled viable tissue area, and record excluded necrotic or damaged regions (standard IHC practice). Score membrane-associated signal separately from cytoplasmic and nuclear signal because full-length ALK is membrane anchored, while the HPA tissue profile also reports cytoplasmic and nuclear staining with a specificity warning (UniProt Q9UM73 topology; HPA tissue IHC: profile and reliability description).
When is an ALK-positive chromogenic result convincing?
A convincing result combines staining in an expected cell population with a reproducible compartment pattern and clean negative controls (standard IHC practice; UniProt Q9UM73 localisation). HPA reports high staining in cerebellar Purkinje cells and cortical and hippocampal neuronal cells, while UniProt describes ALK expression in brain and CNS (HPA tissue IHC: positive cells; UniProt Q9UM73 tissue specificity). Scrutinise isolated nuclear staining, tissue-edge enhancement, necrotic areas and signal reproduced in a no-primary control; endogenous peroxidase can also produce misleading DAB colour (UniProt Q9UM73 topology; standard IHC practice). Interpret unexpected widespread positivity conservatively because HPA notes low staining–RNA consistency and presumed off-target binding; confirm consequential findings with an independent assay (HPA tissue IHC: reliability description; standard IHC practice).
Boster reagents
Best ALK / ALK tyrosine kinase receptor IHC Antibodies
The catalog lists human-reactive ALK antibodies for IHC and IF; one also lists mouse and rat reactivity (catalog: applications and reactivity). The supplied figures show IHC staining only (catalog: image captions).
Real IHC data
Anti-ALK Rabbit Monoclonal Antibody
Cat # M00301-5
Real IHC data
Anti-ALK Rabbit Monoclonal Antibody
Cat # M00301-3
M00301-5 lists human IHC and IF applications and shows IHC staining of human non-Hodgkin lymphoma and neuroblastoma (catalog: applications, reactivity, IHC image captions). M00301-3 lists human, mouse and rat IHC, ICC and IF applications; its IHC figure shows a paraffin-embedded human kidney section (catalog: applications, reactivity, IHC image caption).
Which to pick: For human tissue IHC, choose M00301-5 when lymphoma or neuroblastoma staining is relevant (M00301-5: IHC image captions); choose M00301-3 for a paraffin-section example in human kidney (M00301-3: IHC image caption). For IF/ICC or listed mouse and rat reactivity, choose M00301-3 (M00301-3: applications and reactivity); its supplied figure supports paraffin-section IHC, and the fixative is unreported (M00301-3: IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UM73 (ALK_HUMAN, ALK tyrosine kinase receptor).
- Human Protein Atlas. ALK tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ALK subcellular location (ICC-IF): Localized to the plasma membrane..
- Human Protein Atlas. ALK antibody validation summary (1 antibodies).
- ALK-Positive Renal Cell Carcinoma in a Large Series of Consecutively Resected Korean Renal Cell Carcinoma Patients. Korean journal of pathology 2013 — PMC3830992.
- Anaplastic lymphoma kinase (ALK)-expressing Lung Adenocarcinoma with Combined Neuroendocrine Component or Neuroendocrine Transformation: Implications for Neuroendocrine Transformation and Response to ALK-tyrosine Kinase Inhibitors. Journal of Korean medical science 2018 — PMC5890087.
- Inflammatory Myofibroblastic Tumor: An Updated Review. Cancers 2025 — PMC12026078.
- Colorectal Adenocarcinomas Harboring ALK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 12 Cases and Review of the Literature. The American journal of surgical pathology 2020 — PMC9440614.
- PubMed PMID:9174053 — UniProt-cited evidence.
- PubMed PMID:9053841 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ALK (ALK tyrosine kinase receptor). Expected localisation Membrane expected (UniProt); cytoplasm and nuclei observed (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M00301-5 at 1:200-1:1000 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Purkinje 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 M00301-5); 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-ALK, 1:200-1:1000 (datasheet M00301-5); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target binding may mimic ALK staining (HPA tissue IHC); Isoform / epitope: No isoforms listed; extracellular versus cytoplasmic epitope matters (UniProt); Regulation: Transient in select neural regions (UniProt). Evidence: UniProt Q9UM73, HPA tissue IHC · Approved, HPA ICC-IF · plasma membrane., Datasheet M00301-5, PMC5890087. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ALK/immunohistochemistry-ihc-guide.