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AFAP1L2 / Actin filament-associated protein 1-like 2 · IHC design guide
Design Immunohistochemistry for AFAP1L2
Plan AFAP1L2 chromogenic IHC around the cytoplasmic connective tissue staining reported in spleen and thyroid (HPA tissue IHC). Compare thyroid glandular cells, reported as high, with adipocytes, reported as not detected, while accounting for low agreement between staining and RNA data (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AFAP1L2 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 in spleen and thyroid connective tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in spleen and thyroid connective tissue (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Thyroid gland+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 sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining; low agreement with RNA data (HPA tissue IHC) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | 4 isoforms; effects on the epitope are unknown (UniProt) |
Section 1
Recommended AFAP1L2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with the published cholangiocarcinoma tissue microarray protocol (PMC8592414).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A03350); 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-AFAP1L2, 2.5 μg/mL (datasheet A03350) |
| 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 | AFAP1L2-positive staining in glandular cells of thyroid gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in connective tissue of spleen and thyroid gland. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule; UniProt: cytoplasm). The article does not specify retrieval conditions (PMC8592414).
Section 2
What Is the Expected AFAP1L2 Staining Pattern?
AFAP1L2 is cytoplasmic and has no transmembrane segment (UniProt Q8N4X5: localization and topology). In IHC, expect cytoplasmic staining in thyroid glandular cells and selected other cells; HPA also describes connective tissue staining in spleen and thyroid (HPA: tissue IHC). Treat the pattern as provisional: HPA rates its IHC evidence Approved but reports low staining–RNA consistency and presumed off-target binding (HPA: reliability description).
| Thyroid glandular cells show clear cytoplasmic chromogen, with surrounding tissue architecture visible. | This is the strongest listed cell-level reference: High glandular-cell staining (HPA: thyroid IHC). Score the cytoplasm of identifiable cells, not the whole tissue indiscriminately (general IHC practice). |
| Cytoplasmic signal appears in cerebellar granular-layer cells, kidney glomerular cells, or parathyroid glandular cells. | These are plausible additional positives, each listed at Medium staining (HPA: tissue IHC). Their lower reported level makes them less decisive than thyroid glandular cells when checking a weak run. |
| The dominant signal is nuclear, or it follows a crisp membrane outline without convincing cytoplasm. | A nuclear-dominant pattern conflicts with cytoplasmic localization (UniProt Q8N4X5; HPA: tissue IHC). HPA reports plasma-membrane localization as uncertain in ICC-IF, so membrane signal alone cannot establish a correct IHC result (HPA: subcellular). |
| Strong color fills cells listed as unstained, or a uniform haze obscures many cell types. | Adipocytes and bone-marrow hematopoietic cells are listed as not detected (HPA: tissue IHC). Unexpected cell staining warrants an off-target or endogenous-enzyme check; a broad haze also warrants a background-control check (HPA: reliability description; general IHC practice). |
| No interpretable signal appears in thyroid glandular cells. | This conflicts with the listed High thyroid staining (HPA: thyroid IHC). First check whether the positive-control section and detection run worked; a failed control cannot distinguish technical failure from a negative test specimen (general IHC practice). |
💡Expected AFAP1L2 appearanceCall positive a cell-resolved cytoplasmic stain in thyroid glandular cells, potentially High in the HPA reference; nuclear dominance or broad staining of listed negative cells is suspect (HPA: thyroid IHC and reliability description; UniProt Q8N4X5: cytoplasm).
| Tissue and cell choice | Thyroid glandular cells provide a listed High reference, while adipocytes are listed as not detected (HPA: tissue IHC). Compare named cell types within preserved tissue architecture (general IHC practice). |
| Antibody evidence | The listed rabbit polyclonal antibody HPA037843 is IHC Approved, and HPA reports low staining–RNA consistency plus presumed off-target binding (HPA: antibody record and reliability description). Resolve unexpected patterns with controls rather than intensity alone (general IHC practice). |
| Isoforms and epitope | UniProt lists 4 isoforms and two PH domains (UniProt Q8N4X5). The supplied record does not locate the catalog antibody's epitope, so isoform coverage and domain-dependent staining cannot be predicted from it. |
| Protein topology and processing | AFAP1L2 has no transmembrane segment or signal peptide and is annotated as one chain spanning residues 1–818 (UniProt Q8N4X5). These annotations support a cytoplasmic expectation but do not establish a shedding pattern or a fixation effect. |
| Retrieval and chromogenic detection | No AFAP1L2-specific retrieval condition is supplied. Check retrieval, blocking, detection reagents and counterstain against the run controls as general paraffin-section IHC practice; do not infer target-specific fixation sensitivity from HPA staining levels. |
| IF/ICC Q: What localization should be checked? | A: Look primarily for cytosol; plasma-membrane localization is uncertain (HPA: subcellular ICC-IF). HPA lists images from A-431 and A-549 and rates HPA037843 ICC as Supported (HPA: subcellular and antibody record). |
| Situation | Likely cause | Next action |
|---|---|---|
| Thyroid glandular cells and the run's positive control are both blank. | A shared staining or detection failure is possible (general IHC practice); HPA lists thyroid glandular cells as High (HPA: thyroid IHC). | Confirm section integrity and the run controls, then review retrieval, primary-antibody application and chromogenic detection records (general IHC practice). |
| Thyroid is weak, but several lower-level tissues look equally dark. | A flat intensity pattern conflicts with HPA's High thyroid and Medium cerebellar granular-layer or glomerular references (HPA: tissue IHC). | Compare the named cells at the same staining exposure and score their cytoplasmic signal; examine unexpected cells with a control section (general IHC practice). |
| Nuclei dominate the signal. | Nuclear dominance conflicts with the annotated cytoplasmic location (UniProt Q8N4X5; HPA: tissue IHC). | Check whether counterstain or precipitate is being mistaken for specific chromogen, and compare with the no-primary control (general IHC practice). |
| Adipocytes or bone-marrow hematopoietic cells stain strongly. | Both are listed as not detected; HPA also notes presumed off-target binding (HPA: tissue IHC and reliability description). Endogenous detection activity is another general IHC possibility. | Review cell identity and use no-primary and appropriate detection controls to investigate background or endogenous activity before calling AFAP1L2 positive (general IHC practice). |
| Diffuse brown haze hides cell borders. | Non-specific reagent binding or inadequate background control may obscure localization (general IHC practice); diffuse haze cannot establish HPA's cell-specific pattern (HPA: tissue IHC). | Inspect no-primary and detection controls, then review blocking, washing, chromogen development and counterstain; score only resolved cellular staining (general IHC practice). |
| A sharp membrane rim is the only reproducible signal. | HPA's plasma-membrane ICC-IF annotation is uncertain, whereas its main location is supported cytosol (HPA: subcellular); UniProt lists no transmembrane segment (UniProt Q8N4X5). | Seek accompanying cytoplasmic staining in the listed positive cells and compare control sections before interpreting a membrane-only IHC pattern (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing AFAP1L2 staining
A03350 · IHCImmunohistochemistry of AFAP1L2 in human liver tissue with AFAP1L2 antibody at 2.5 μg/mL.
A03350 · IFImmunofluorescence of AFAP1L2 in human liver tissue with AFAP1L antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169129-AFAP1L2/tissue/cerebral+cortex#imid_9315440
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000169129-AFAP1L2/tissue/thyroid+gland#imid_9315392
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169129-AFAP1L2/tissue/colon#imid_9285854
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169129-AFAP1L2/tissue/liver#imid_9285855
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037843; green = AFAP1L2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037843; green = AFAP1L2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AFAP1L2 IHC & IF
🧪Run thyroid gland first: its glandular cells should stain (HPA: thyroid glandular cells High). Use adipose tissue as the negative tissue: adipocytes should show background staining (HPA: adipocytes Not detected); the supplied HPA rows do not identify a verified internal negative cell type within thyroid, so assess any unstained cells on that slide against the no-primary control (HPA: thyroid glandular cells High; standard IHC practice).
Positive control tissue: Thyroid gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AFAP1L2 in A-431, A-549, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-matched, isotype-matched antibody controls; use AFAP1L2 knockout tissue or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). Block endogenous peroxidase for chromogenic detection and check thyroid colloid for background signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval on matched paraffin sections (supplied AFAP1L2 evidence; standard IHC practice). The selected A03350 human liver IHC caption reports 2.5 μg/mL, but its fixative is unreported (selected A03350 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF/ICC are easier for AFAP1L2; check thyroid colloid for nonspecific signal when interpreting the positive slide (supplied AFAP1L2 evidence; standard IHC practice).
HPA tissue IHC evidence for AFAP1L2
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. 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 |
|---|---|---|---|---|
| Thyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Medium | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AFAP1L2 IHC Tips
Troubleshoot AFAP1L2 staining in paraffin sections by checking retrieval, compartment, cell identity and controls before comparing chromogenic signal across specimens.
Which retrieval conditions should I start with for AFAP1L2 paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced epitope retrieval at 95–98 °C for 20 min (page antigen retrieval rule). If staining is weak, compare a second retrieval condition on matched sections while holding antibody concentration and detection conditions constant (standard IHC practice). Excessive retrieval can damage morphology or raise diffuse background, so assess tissue preservation alongside signal (standard IHC practice). The catalog image reports AFAP1L2 staining in human liver at 2.5 µg/mL, but its caption does not state retrieval conditions or fixative (A03350 tissue IHC caption).
How should I investigate weak AFAP1L2 staining after fixation?
AFAP1L2 specific sensitivity to fixation is unknown from the supplied evidence, so treat fixation time as a variable to test rather than an established cause of weak staining. Compare sections with documented, consistent fixation and processing histories before changing retrieval or antibody concentration (standard IHC practice). On a control section, confirm that morphology is preserved and that cytoplasmic staining can be distinguished from diffuse deposit (UniProt Q8N4X5: cytoplasm; standard IHC practice). The liver image caption supplies an antibody concentration of 2.5 µg/mL but does not identify a fixative (A03350 tissue IHC caption).
Should AFAP1L2 staining appear in the cytoplasm or at the cell surface?
Use cytoplasmic staining as the principal pattern when evaluating AFAP1L2 in paraffin sections (UniProt Q8N4X5: cytoplasm; HPA subcellular: supported cytosol). HPA also reports plasma membrane localisation with uncertain support, so score a membrane rim separately from cytoplasmic signal (HPA subcellular: plasma membrane uncertain). AFAP1L2 has no transmembrane segment, and its membrane association should not be interpreted as evidence of a membrane spanning protein (UniProt Q8N4X5 topology). Compare compartments within the same cell population and inspect a counterstained section for tissue edges, pigment and precipitate before assigning a new pattern (standard IHC practice).
Could isoforms or phosphorylation change what my IHC antibody detects?
AFAP1L2 has 4 listed isoforms and 2 PH domains, so establish which sequence region the antibody recognizes before interpreting differences among specimens (UniProt Q8N4X5). The record includes phosphorylation at Tyr56, Ser408, Tyr413 and Ser484; whether any modification affects this antibody's epitope is unknown (UniProt Q8N4X5). Compare the immunogen sequence with isoform sequences when available, and avoid claiming isoform specificity from staining alone (standard IHC practice). If an epitope spans a splice junction or modified residue, confirm the interpretation with an independently characterized antibody or orthogonal expression evidence (standard IHC practice).
How can IF help check an ambiguous AFAP1L2 IHC pattern?
Use IF as a separate check of cell identity and compartment when chromogenic AFAP1L2 staining is ambiguous (standard IHC and IF practice). For thyroid sections, multiplex AFAP1L2 with a glandular cell marker and assess whether the signals occupy the same cells (HPA tissue IHC: high in thyroid glandular cells). Select a fluorophore channel with low tissue autofluorescence and include an unstained section to measure that background (standard IF practice). Because AFAP1L2 is principally cytosolic and has no transmembrane segment, use a validated permeabilisation condition for access to an intracellular epitope; the IHC caption provides no IF fixation or permeabilisation method (HPA subcellular: supported cytosol; UniProt Q8N4X5 topology; A03350 tissue IHC caption).
What should I check when AFAP1L2 DAB staining looks diffuse?
First inspect a no primary antibody section and the hematoxylin counterstain to distinguish diffuse DAB deposit from cellular signal (standard IHC practice). In a peroxidase based workflow, apply an endogenous peroxidase block and check whether residual enzyme activity follows blood rich or damaged areas (standard IHC practice). Reassess blocking, antibody concentration and wash stringency on matched sections; 2.5 µg/mL is the concentration reported for the catalog liver image, not a universal working concentration (A03350 tissue IHC caption). Interpret unexpected widespread staining cautiously because HPA reports low agreement between antibody staining and RNA data and notes presumed off target binding (HPA tissue IHC reliability description).
How should I score AFAP1L2 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, then use an H-score based on staining intensity and the percentage of positive cells (UniProt Q8N4X5: cytoplasm; standard IHC practice). Record the percentage of positive target cells separately so a change in cell abundance is distinguishable from a change in staining intensity (standard IHC practice). For spatial comparisons, count positive cells per mm² and normalize to the area of evaluable tissue or the number of target cells (standard IHC practice). Keep retrieval, exposure to chromogen and scoring thresholds consistent across sections, and report membrane staining separately because its localisation support is uncertain (HPA subcellular: plasma membrane uncertain; standard IHC practice).
How do I distinguish genuine AFAP1L2 signal from staining artefact?
Favor reproducible cytoplasmic signal in anatomically identified cells over a nuclear only pattern or staining confined to section edges (UniProt Q8N4X5: cytoplasm; standard IHC practice). Thyroid glandular cells provide a reported high staining population, whereas liver cholangiocytes are reported as not detected by HPA (HPA tissue IHC). Check necrotic regions, pigment and no primary controls before interpreting dense DAB or endogenous peroxidase activity as AFAP1L2 (standard IHC practice). The catalog liver image documents staining at 2.5 µg/mL, but that caption does not establish which liver cells stained; HPA also flags presumed off target binding and low agreement with RNA (A03350 tissue IHC caption; HPA tissue IHC reliability description).
Boster reagents
Best AFAP1L2 / Actin filament-associated protein 1-like 2 IHC Antibodies
A03350 has IHC and IF images from human liver tissue (catalog image captions); its listed reactivity covers human, mouse, and rat (catalog: reactivity).
Real IHC data
Anti-AFAP1L2 Antibody
Cat # A03350
A03350 will render with an IHC image of human liver tissue at 2.5 μg/mL (A03350 IHC image caption). The catalog also lists IHC-P and IF applications and shows IF in human liver tissue at 20 μg/mL (catalog: applications; A03350 IF image caption).
Which to pick: Choose A03350 for paraffin-section IHC: IHC-P is listed and its own human liver IHC image shows staining at 2.5 μg/mL; the fixative is unreported (catalog: applications; A03350 IHC image caption). A03350 is also the IF option at 20 μg/mL in human liver tissue, while ICC requires separate validation (catalog: applications; A03350 IF image caption). For mouse or rat samples, A03350 lists reactivity with both species, but its supplied IHC and IF images show human tissue; it is a rabbit antibody with no clone reported (catalog: reactivity, host and clone; A03350 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N4X5 (AF1L2_HUMAN, Actin filament-associated protein 1-like 2).
- Human Protein Atlas. AFAP1L2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. AFAP1L2 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane..
- Human Protein Atlas. AFAP1L2 antibody validation summary (1 antibodies).
- Adaptor protein XB130 regulates the aggressiveness of cholangiocarcinoma. PloS one 2021 — PMC8592414.
- PubMed PMID:11572484 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AFAP1L2 (Actin filament-associated protein 1-like 2). Expected localisation Cytoplasm in spleen and thyroid connective tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03350 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Thyroid gland (Glandular 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 A03350); 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-AFAP1L2, 2.5 μg/mL (datasheet A03350); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining; low agreement with RNA data (HPA tissue IHC); Isoform / epitope: 4 isoforms; effects on the epitope are unknown (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q8N4X5, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet A03350, PMC8592414. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AFAP1L2/immunohistochemistry-ihc-guide.