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AFAP1L2 / Actin filament-associated protein 1-like 2 · IHC design guide
Design Immunohistochemistry for AFAP1L2
Plan AFAP1L2 IHC on paraffin sections using the catalog antibody at a starting concentration of 2.5 μg/mL (datasheet: IHC-P). Assess cytoplasmic staining in spleen and thyroid connective tissue (HPA tissue IHC), and interpret unexpected staining in light of presumed off-target binding (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 | Cytoplasmic 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining; low RNA–IHC consistency (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 4 isoforms; cytoplasmic target, epitope coverage unknown (UniProt) |
Section 1
Recommended AFAP1L2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published AFAP1L2/XB130 protocol for cholangiocarcinoma tissue microarrays (PMC8592414: methods).
| 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 retrieval at 95–98 °C for 20 min (page antigen retrieval; UniProt Q8N4X5: cytoplasm).
Section 2
What Is the Expected AFAP1L2 Staining Pattern?
AFAP1L2 is a cytoplasmic protein with no transmembrane segment (UniProt Q8N4X5 localization and topology). In IHC, expect cytoplasmic staining in thyroid glandular cells and in connective tissue of the thyroid and spleen (HPA tissue IHC). Interpret these patterns cautiously: HPA rates the tissue antibody Approved but reports low consistency with RNA data and presumed off-target staining that was disregarded (HPA tissue IHC reliability).
| Cytoplasmic staining in thyroid glandular cells. | This matches an HPA-listed High cell-level result and UniProt's cytoplasmic localization (HPA tissue IHC; UniProt Q8N4X5). HPA also describes cytoplasmic expression in thyroid connective tissue; assess glandular and connective tissue cells separately rather than treating every stained area as the same finding (HPA tissue IHC profile). |
| Predominantly nuclear staining, with little cytoplasmic signal. | This does not match the reported cytoplasmic location (UniProt Q8N4X5; HPA tissue IHC). Check whether the color lies within nuclei or overlaps the cytoplasm before interpreting it as AFAP1L2; staining in an unexpected compartment can reflect nonspecific detection or a slide artifact (general IHC practice). |
| Strong staining in a cell type HPA lists as not detected. | For example, adipocytes or cardiomyocytes are listed as Not detected (HPA tissue IHC). Such staining warrants a specificity check, including the possibility of cross-reactivity or endogenous detection activity (general IHC practice). HPA reports presumed off-target staining in its tissue assessment, so an isolated unexpected positive needs caution (HPA tissue IHC reliability). |
| Diffuse color across cells and surrounding tissue. | A broad deposit that does not follow cell boundaries is difficult to score as cytoplasmic AFAP1L2 (UniProt Q8N4X5 localization; general IHC practice). Compare tissue morphology and a control slide; inadequate blocking, residual detection activity, or excess chromogen development can produce background (general IHC practice). |
| No discernible signal in thyroid glandular cells. | HPA records High staining in those cells, so first review tissue identity, preserved morphology, controls, and the antibody's IHC-P workflow (HPA tissue IHC; general IHC practice). Absence on one slide is not proof that AFAP1L2 is absent: the HPA tissue assessment is Approved but has low antibody–RNA consistency (HPA tissue IHC reliability). |
💡Expected AFAP1L2 appearanceCall positive IHC when color resolves in the cytoplasm of identifiable thyroid glandular cells, where HPA reports High staining; HPA also reports cytoplasmic thyroid and spleen connective tissue expression, while isolated nuclear color or strong staining in HPA-negative cell types is suspect (HPA tissue IHC; UniProt Q8N4X5; general IHC practice).
| Cell type and tissue | Expected intensity depends on the annotated cells: thyroid glandular cells are High; kidney glomerular cells and cerebellar granular-layer cells are Medium; adipocytes and cardiomyocytes are Not detected (HPA tissue IHC). Identify the cell type before scoring a section (general IHC practice). |
| Localization and topology | UniProt places AFAP1L2 in the cytoplasm and reports no transmembrane segment (UniProt Q8N4X5). A dominant nuclear-only IHC pattern therefore needs review; topology alone cannot establish how an antibody epitope behaves in a paraffin section (UniProt Q8N4X5; general IHC practice). |
| Antibody evidence | The listed antibody, HPA037843, is IHC Approved, while the tissue assessment notes low consistency with RNA expression and disregarded presumed off-target staining (HPA antibodies; HPA tissue IHC reliability). Use the reported pattern as a reference, with controls for unexpected positives (general IHC practice). |
| IF/ICC: where should fluorescence appear? | Primarily in the cytosol: HPA marks that location Supported and reports plasma membrane localization as Uncertain (HPA subcellular ICC-IF). Membrane fluorescence alone is therefore a weaker localization call; the ICC result does not specify an IHC-P staining intensity (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Thyroid glandular cells have no visible stain. | The section or detection run may have failed, or the cells may have been misidentified; HPA reports High staining for these cells (HPA tissue IHC; general IHC practice). | Confirm glandular-cell morphology and review the run's positive and detection controls. Check each IHC-P step against the catalog antibody's instructions before changing conditions (general IHC practice). |
| Color is confined mainly to nuclei. | A nuclear-only pattern conflicts with the reported cytoplasmic localization (UniProt Q8N4X5; HPA tissue IHC). Counterstain overlap or nonspecific deposition may complicate reading (general IHC practice). | Inspect the nuclear and cytoplasmic boundaries at higher magnification and compare a control slide; score AFAP1L2 only where a cellular cytoplasmic pattern is convincing (general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly. | Both cell types are listed as Not detected (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA also notes presumed off-target staining (HPA tissue IHC reliability). | Verify cell identity and compare an appropriate negative control. Review endogenous-activity blocking for the chosen detection system before assigning biological meaning to the stain (general IHC practice). |
| Diffuse background obscures cell boundaries. | Broad color deposition can arise from insufficient blocking, incomplete washing, or excessive chromogen development (general IHC practice). It does not give a scorable cytoplasmic pattern (UniProt Q8N4X5; general IHC practice). | Compare a negative control, inspect wash and blocking steps, and review development time for the detection system; reassess only where cells remain individually resolvable (general IHC practice). |
| A weak signal appears in a tissue expected to be positive. | HPA reports different levels by cell type: kidney glomerular cells are Medium, while thyroid glandular cells are High (HPA tissue IHC). Sampling or cell identification may account for an apparent mismatch (general IHC practice). | Identify the annotated cells before comparing intensity. Review tissue morphology and run controls, then report the observed compartment and strength rather than assigning one expected level to an entire organ (HPA tissue IHC; general IHC practice). |
| Membrane-edge color is interpreted as the main positive pattern. | The HPA ICC-IF summary supports cytosol and labels its additional plasma membrane location Uncertain (HPA subcellular ICC-IF). A membrane-only IHC call exceeds that localization evidence (HPA subcellular ICC-IF; UniProt Q8N4X5). | Recheck whether cytoplasmic color is present in the same cells and compare the negative control. Record membrane-edge color separately if it persists, without counting it alone as the expected IHC pattern (general IHC practice; HPA subcellular ICC-IF). |
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: glandular cells should stain (HPA: High in thyroid gland glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the thyroid slide, assess cells outside the glandular compartment for background, without treating them as a validated negative population (HPA: High is assigned to glandular cells).
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 a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host species, clonality and concentration, and, where available, AFAP1L2 knockout material as a biological negative (standard IHC practice). Check the thyroid section for endogenous peroxidase signal before chromogenic detection; if using a biotin-based system, check endogenous biotin background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected A03350 tissue-IHC caption gives 2.5 μg/mL for human liver but does not report a fixative (A03350 caption). Antigen-retrieval dependence and whether frozen sections or IF are easier are likewise unreported, so establish retrieval conditions empirically on the positive tissue (standard IHC practice; HPA: High in thyroid gland glandular cells). Thyroid endogenous peroxidase can complicate chromogenic interpretation; evaluate it with the no-primary control (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 by checking retrieval, compartment, cell identity, and controls before comparing signal across sections.
How should I retrieve AFAP1L2 in paraffin sections if cytoplasmic staining is weak?
Use citrate pH 6.0 heat induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Keep section thickness, cooling, and antibody incubation consistent when comparing retrieval runs (standard IHC practice). If signal remains weak, compare a modestly longer retrieval with the stated condition on adjacent sections, watching for tissue damage or increased background (standard IHC practice). Score cytoplasmic signal in a documented positive reference region, such as thyroid glandular cells, and include a negative reagent control; thyroid glandular staining is reported as high, but the HPA profile carries a consistency caveat (HPA: High in thyroid glandular cells; HPA: reliability description).
Could fixation explain inconsistent AFAP1L2 staining between paraffin blocks?
Target specific fixation sensitivity is unknown because no AFAP1L2 fixation comparison is supplied (supplied evidence: no target specific fixation study). Record the fixative, fixation duration, processing schedule, and block age before attributing a difference to the antigen (standard IHC practice). Compare adjacent sections under the same citrate pH 6.0, 95–98 °C, 20 min retrieval and detection conditions, then assess morphology alongside staining (page retrieval rule; standard IHC practice). The A03350 liver image caption reports 2.5 µg/mL antibody but does not state its fixative, so it cannot establish fixation tolerance for that image or other blocks (A03350 tissue IHC caption).
Should AFAP1L2 staining appear in cytoplasm, membrane, or nucleus?
Prioritise cytoplasmic staining when reading chromogenic sections: AFAP1L2 is annotated as cytoplasmic, and the supported HPA subcellular location is cytosol (UniProt Q8N4X5: subcellular location; HPA subcellular: supported cytosol). A membrane accent can be investigated, but HPA labels plasma membrane localisation uncertain and UniProt reports no transmembrane segment (HPA subcellular: uncertain plasma membrane; UniProt Q8N4X5: topology). Compare the suspected compartment with a haematoxylin counterstain and inspect several well preserved cells away from section edges (standard IHC practice). Predominantly nuclear or extracellular brown signal warrants checks for nonspecific detection and tissue artefact before it is assigned to AFAP1L2 (UniProt Q8N4X5: subcellular location; standard IHC practice).
How do isoforms and phosphorylation affect interpretation of this antibody's IHC signal?
AFAP1L2 has 4 annotated isoforms and 2 PH domains, spanning residues 175–271 and 353–447 (UniProt Q8N4X5: isoforms and domains). Its recorded modifications include phosphotyrosines at 56 and 413, plus phosphoserines at 408 and 484 (UniProt Q8N4X5: modified residues). Without a supplied epitope map, do not assign an IHC difference to a particular isoform or phosphorylation state (supplied evidence: no epitope map; standard IHC interpretation). Ask for epitope and isoform coverage, then compare independently validated antibodies or orthogonal expression evidence on matched regions before making that claim (standard IHC practice).
How can IF help resolve ambiguous AFAP1L2 staining seen by chromogenic IHC?
On a separate IF/ICC experiment, multiplex AFAP1L2 with a marker that identifies thyroid glandular cells when evaluating that tissue; HPA reports high AFAP1L2 staining there (HPA: High in thyroid glandular cells; standard IF practice). Select spectrally separated fluorophores after checking tissue autofluorescence, and include single colour and unstained controls to assess bleed through and native fluorescence (standard IF practice). For an intracellular epitope, use controlled permeabilisation because the supported location is cytosol and AFAP1L2 has no transmembrane segment (HPA subcellular: supported cytosol; UniProt Q8N4X5: topology; standard IF practice). Compare compartment and cell identity across methods without assuming identical signal intensity or an established antibody specific IF protocol (standard IHC/IF practice; supplied evidence: no IF protocol).
What should I check when the whole section develops brown background?
Inspect the no primary control alongside the stained section to separate detection chemistry from antibody dependent background (standard chromogenic IHC practice). Confirm endogenous peroxidase blocking before DAB development, and review blocking, washes, reagent concentration, and development time using matched sections (standard chromogenic IHC practice). If background rises after retrieval, repeat the specified citrate pH 6.0, 95–98 °C, 20 min condition before changing another variable (page retrieval rule; standard IHC practice). Use the 2.5 µg/mL liver caption only as a record of that A03350 image, since it does not define a universal working concentration or explain diffuse staining (A03350 tissue IHC caption).
How should I quantify AFAP1L2 across sections with mixed cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Predefine the compartment and cell population to score, favouring cytoplasmic signal because that is the supported AFAP1L2 location (HPA subcellular: supported cytosol; standard IHC practice). Within matched tissue regions, report an H-score from staining intensity and the percentage of positive target cells, or report positive cells per mm² when counting discrete cells (standard IHC quantification practice). Normalise counts to evaluable target cells or viable tissue area, and apply the same threshold, counterstain review, and imaging settings across samples (standard IHC quantification practice). Record cell type separately from tissue level because HPA reports high staining in thyroid glandular cells while its antibody staining and RNA data have low consistency (HPA: High in thyroid glandular cells; HPA: reliability description).
How can I distinguish genuine AFAP1L2 positivity from staining artefact?
Treat reproducible cytoplasmic signal in morphologically intact cells as more plausible than isolated nuclear or extracellular deposits, given the supported cytosolic location (HPA subcellular: supported cytosol; standard IHC interpretation). Check whether the stained cells match the annotation: HPA reports high thyroid glandular staining but no detectable signal in liver cholangiocytes (HPA: thyroid glandular cells High; HPA: liver cholangiocytes Not detected). Exclude section edge enhancement, necrotic regions, and endogenous enzyme activity with morphology review, a no primary control, and an appropriate peroxidase block (standard chromogenic IHC practice). Interpret disagreements cautiously because HPA reports low agreement between antibody staining and RNA expression and notes presumed off target binding (HPA: 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 is listed for IHC-P and IF (catalog: applications). Its IHC and IF images each show human liver tissue (catalog: A03350 image captions).
Which to pick: Choose A03350 for tissue IHC: it is listed for IHC-P, and its own IHC caption shows human liver tissue stained at 2.5 μg/mL (catalog: applications; A03350 IHC image caption). The same SKU is listed for IF, with a human liver tissue image at 20 μg/mL; ICC-specific validation is unreported (catalog: applications; A03350 IF image caption). Its listed reactivity includes human, mouse, and rat, but the supplied IHC and IF images show human tissue only; the IHC fixative is unreported, and clonality is unspecified (catalog: reactivity; A03350 image captions; catalog: clone).
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 Cytoplasmic 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 paraffin sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining; low RNA–IHC consistency (HPA tissue IHC); Isoform / epitope: 4 isoforms; cytoplasmic target, epitope coverage unknown (UniProt); Regulation: No expression regulator specified (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.