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FLG / Filaggrin · IHC design guide
Design Immunohistochemistry for FLG
Plan chromogenic FLG IHC in paraffin sections using skin’s granular layer as a strong positive reference (HPA tissue IHC). This guide covers staining setup and interpretation of the superficial cytoplasmic pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FLG 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 | Superficial squamous-cell cytoplasm (HPA tissue IHC) | |
| Staining pattern | Granular-layer cells show selective cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9870) | |
| Positive control | Skin+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 | Signal depends on the proportion of granular cells (HPA tissue IHC) | |
| Regulation | Granular-layer enrichment (HPA tissue IHC) | |
| Isoform / epitope | No isoforms annotated; one full-length chain (UniProt) |
Section 1
Recommended FLG IHC & IF Protocols
The catalog antibody protocol uses heat-mediated EDTA pH 8.0 retrieval (datasheet: PB9870). The published FLG IHC protocols below cover mouse, guinea pig, and human skin (PMC10272903; PMC7859006; PMC9489357; PMC12672505).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet PB9870) |
| Fixation | Image fixative and duration unreported (datasheet PB9870); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet PB9870); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9870) |
| Primary antibody | Rabbit anti-FLG, 0.5-1μg/ml (datasheet PB9870) |
| Primary incubation | Overnight at 4 °C (datasheet PB9870) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9870) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FLG-positive staining in cells in granular layer of skin (HPA tissue IHC: High). HPA tissue profile: Highly selective cytoplasmic expression in superficial layers in squamous epithelia. No signal in the no-primary control. |
💡Decision noteFor the catalog antibody, start with heat-mediated EDTA pH 8.0 retrieval (datasheet: PB9870); the cited articles do not specify a retrieval buffer (PMC10272903; PMC7859006; PMC9489357; PMC12672505).
Section 2
What Is the Expected FLG Staining Pattern?
FLG should stain the cytoplasm of keratinocytes in the skin’s granular layer, where it localizes to keratohyalin granules (UniProt P20930). HPA reports high staining in these cells and selective cytoplasmic staining in superficial squamous epithelia (HPA tissue IHC). FLG has no transmembrane segment (UniProt P20930 topology). HPA rates the tissue IHC profile Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Strong cytoplasmic staining is concentrated in the skin’s granular layer. | This matches the reported high granular-layer signal (HPA tissue IHC) and keratohyalin-granule localization (UniProt P20930). Judge the spatial pattern alongside intensity: a convincing positive follows the superficial differentiation layer, rather than appearing evenly across the epidermis. |
| Superficial squamous cells stain in esophagus, oral mucosa, tonsil, or vagina. | Medium staining is reported for squamous epithelial cells at each of these sites (HPA tissue IHC). A weaker result than the high skin granular-layer signal can therefore fit the reference pattern; compare the correct cell layer and cytoplasmic compartment before calling it negative. |
| The strongest signal is nuclear or confined to cell membranes. | That distribution conflicts with cytoplasmic staining in superficial squamous epithelia (HPA tissue IHC) and FLG’s granule localization and lack of a transmembrane segment (UniProt P20930). Treat it as suspect staining and check detection controls and the antibody’s IHC validation. |
| Cells outside the expected squamous layer stain, or a reported negative cell population stains strongly. | Assess cross-reactivity or endogenous detection activity before assigning FLG expression (general IHC practice). For example, HPA reports no staining in adrenal glandular cells or bronchial respiratory epithelial cells (HPA tissue IHC); those are useful comparisons when present in the study. |
| The slide has diffuse background, or skin granular-layer cells show no signal. | Diffuse color obscures the expected selective cytoplasmic pattern (HPA tissue IHC); review background controls and staining conditions (general IHC practice). No signal in a known-positive skin section calls for a run-level check before interpreting an unknown sample as FLG-negative (HPA tissue IHC; general IHC practice). |
💡Expected FLG appearanceCall an IHC section positive when high cytoplasmic signal marks skin granular-layer cells, with superficial squamous staining at reported sites; dominant nuclear, membrane-only, or widespread unrelated-cell staining is suspect (HPA tissue IHC; UniProt P20930).
| Tissue and layer selected for assessment | Skin granular-layer cells are the clearest reported positive, with high staining (HPA tissue IHC). Esophageal, oral, tonsillar, and vaginal squamous cells show medium staining; cervical squamous cells show low staining (HPA tissue IHC). Account for those differences when choosing a comparison section. |
| Intracellular pattern used for scoring | FLG localizes to keratohyalin granules in the stratum granulosum and has no transmembrane segment (UniProt P20930). Prioritize cytoplasmic signal in the appropriate superficial cells; a nuclear or membrane-only pattern requires investigation before it is scored as specific staining (HPA tissue IHC; general IHC practice). |
| Antibody evidence and interpretation limit | HPA lists IHC Enhanced status for HPA030188, HPA030189, and CAB002210 (HPA antibodies). Its overall tissue profile is also Enhanced, but antibody staining and RNA expression have only medium consistency (HPA tissue IHC). Interpret unexpected staining with tissue and detection controls. |
| Can the same pattern guide IF/ICC interpretation? | HPA reports FLG in cytoplasmic bodies by ICC-IF, with images from HaCaT cells (HPA subcellular). That observation can inform a localization check, while this page’s expected tissue-layer pattern comes from IHC (HPA tissue IHC). Use the separate IF/ICC guide for assay design. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the skin granular layer | A known-positive cell layer failed to show its reported high signal (HPA tissue IHC); the result may reflect an unsuccessful IHC run (general IHC practice). | Check a skin positive-control section from the same run, then review antigen retrieval, antibody dilution, detection reagents, and counterstain visibility using the validated IHC workflow (general IHC practice). |
| Color covers most cells or extracellular areas | Widespread color does not match selective superficial cytoplasmic expression (HPA tissue IHC); nonspecific staining or detection background is possible (general IHC practice). | Review the negative detection control, blocking and washes, and chromogen development; then compare with the expected skin granular-layer distribution (general IHC practice; HPA tissue IHC). |
| Signal is mainly nuclear or outlines membranes | The compartment conflicts with FLG’s cytoplasmic granule localization (UniProt P20930) and the HPA cytoplasmic tissue profile (HPA tissue IHC). | Inspect the antibody’s IHC validation and detection controls, then rescore only signal in the expected superficial cytoplasm (HPA antibodies; HPA tissue IHC; general IHC practice). |
| Strong staining appears in an HPA-negative cell population | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA reports no staining in adipocytes from adipose tissue or adrenal glandular cells (HPA tissue IHC). | Check a negative detection control and compare the cell identity and compartment with the HPA tissue profile before assigning that signal to FLG (general IHC practice; HPA tissue IHC). |
| A non-skin squamous section looks weaker than skin | HPA reports medium staining in esophageal, oral, tonsillar, and vaginal squamous cells, versus high staining in skin granular-layer cells (HPA tissue IHC). | Score localization and the relevant tissue’s reported level together. Do not require skin-level intensity to recognize the documented medium superficial squamous pattern (HPA tissue IHC). |
| Cervical squamous cells show only faint signal | Low staining is reported for cervical squamous cells (HPA tissue IHC), so intensity alone gives limited confidence in a faint result. | Compare with a same-run skin positive control and a negative detection control; use the cytoplasmic, superficial distribution when deciding whether the cervical signal is credible (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing FLG staining
PB9870 · IHCIHC analysis of Filaggrin using anti-Filaggrin antibody (PB9870). Filaggrin was detected in a paraffin-embedded section of human tonsil tiss
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143631-FLG/tissue/cerebral+cortex#imid_712856
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143631-FLG/tissue/colon#imid_7799858
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143631-FLG/tissue/liver#imid_712775
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000143631-FLG/tissue/kidney#imid_712792
HPA · ICC-IF HaCaTHuman Protein Atlas immunofluorescence, HaCaT, antibody HPA027505; green = FLG, blue = nucleus, red = microtubules.
HPA · ICC-IF HaCaTHuman Protein Atlas immunofluorescence, HaCaT, antibody HPA027505; green = FLG, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FLG IHC & IF
🧪Run skin first: cells in the granular layer should stain strongly (HPA: High in skin granular-layer cells). Use adrenal gland as a negative tissue, with glandular cells showing no specific staining (HPA: Not detected in adrenal glandular cells); on the skin slide, basal epidermal cells should show little or no signal relative to the granular layer (UniProt P20930: expression mainly in the stratum granulosum).
Positive control tissue: Skin (Cells in granular layer, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FLG in HaCaT, with annotated localisation: Cytoplasmic bodies (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit isotype control (selected PB9870 caption: rabbit primary antibody). Use FLG knockout tissue as a biological negative if available; for DAB detection, quench endogenous peroxidase and check endogenous biotin background because the selected PB9870 caption uses biotinylated secondary antibody and a streptavidin–biotin complex.
⚠️Feasibility: The selected PB9870 paraffin-section caption reports EDTA retrieval at pH 8.0 and primary incubation at 1 μg/ml overnight at 4°C, but its fixative is unreported; no target-specific fixation window or fixation effect is reported in the supplied evidence. That caption supports trying its retrieval conditions, but the supplied evidence does not establish that retrieval is essential or that frozen sections or IF are easier. In skin, distinguish granular-layer signal from pigment or nonspecific staining in the keratinized surface (HPA: High in skin granular-layer cells; UniProt P20930: localization in keratohyalin granules).
HPA tissue IHC evidence for FLG
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Skin | Cells in granular layer | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FLG IHC Tips
Use the granular epidermal layer as the main reference for FLG staining, and evaluate tonsil staining in its squamous epithelium (UniProt P20930; HPA tissue IHC).
Which retrieval conditions should I try first for FLG in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9870). The selected tonsil image used that retrieval condition, followed by 10% goat serum block and 1 μg/ml primary antibody overnight at 4°C (caption PB9870). If staining is weak, first check heating consistency and section adherence before changing retrieval conditions, using the granular epidermal layer or tonsillar squamous epithelium to assess the result (standard IHC practice; HPA tissue IHC). Run a matched section without primary antibody when comparing retrieval conditions, because background can otherwise resemble a gain in FLG signal (standard IHC practice).
How should I troubleshoot weak FLG staining when fixation history is uncertain?
The selected image documents a paraffin-embedded tonsil section but does not state its fixative, so target-specific FLG sensitivity to fixation is unknown (caption PB9870). Record the fixative and fixation duration for each specimen, and compare similarly processed sections before attributing weak staining to biological loss (standard IHC practice). Start with the documented EDTA pH 8.0 heat retrieval and 1 μg/ml primary incubation overnight at 4°C, then assess whether the expected epithelial layer is preserved (caption PB9870; HPA tissue IHC). If staining varies with processing history, report that association as an experimental observation rather than an established FLG-specific fixation effect (standard IHC practice).
Where should convincing FLG staining appear in a tissue section?
In skin, look chiefly for cytoplasmic staining in granular-layer keratinocytes, consistent with FLG in keratohyalin granules of the stratum granulosum (HPA tissue IHC; UniProt P20930). In tonsil, evaluate squamous epithelial cells rather than treating all tonsillar cells as equivalent targets; their reported staining is medium, while skin granular-layer staining is high (HPA tissue IHC). Compare the signal with tissue architecture and a counterstain, and score the relevant epithelial compartment separately from adjacent tissue (standard IHC practice; HPA tissue IHC). Diffuse nuclear staining alone would conflict with the reported cytoplasmic localisation and warrants review of controls and detection background (UniProt P20930; HPA tissue IHC; standard IHC practice).
Could FLG processing or epitope placement explain discordant staining?
FLG has no annotated isoforms in the supplied record, and its 4061-residue sequence includes repeated regions; these facts alone do not identify the catalog antibody’s epitope (UniProt P20930). Interpret regional staining cautiously because the record describes a precursor involved in keratin aggregation during terminal epidermal differentiation, while the antibody epitope and its accessibility in sections are unspecified (UniProt P20930; standard IHC practice). If staining differs across superficial layers, compare adjacent sections processed with identical EDTA pH 8.0 retrieval and primary incubation before proposing an epitope explanation (datasheet PB9870; standard IHC practice). Avoid assigning a particular processed fragment or isoform from chromogenic staining without epitope-specific evidence (standard IHC practice).
How can IF help check FLG localisation in a multiplex experiment?
For a separate IF experiment, pair FLG with a granular-layer keratinocyte marker so the expected cell population and FLG distribution can be evaluated together (UniProt P20930; standard IF practice). Choose spectrally separated fluorophores after checking tissue autofluorescence in an unstained section, and include single-label controls to assess bleed-through (standard IF practice). Because FLG is reported in cytoplasmic keratohyalin granules and has no annotated transmembrane segment, use permeabilisation appropriate for an intracellular epitope, then assess whether punctate signal occurs in the expected layer (UniProt P20930; standard IF practice). Keep IF findings distinct from the catalog antibody’s paraffin-section chromogenic result, which documents EDTA pH 8.0 retrieval in tonsil (caption PB9870).
What should I check when FLG DAB staining appears widespread?
The selected tonsil image used a biotinylated secondary, streptavidin-biotin detection and DAB, so examine a matched no-primary section for detection-related staining (caption PB9870; standard IHC practice). Use a peroxidase block and assess whether endogenous enzyme activity contributes to DAB signal; this is a general chromogenic IHC control, not FLG-specific evidence (standard IHC practice). Confirm that the 10% goat serum block and 1 μg/ml primary concentration were applied as documented before changing antibody concentration (caption PB9870). Treat staining outside superficial squamous epithelium cautiously, because the reported tissue profile is highly selective there (HPA tissue IHC).
How should I quantify FLG across differently processed sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the granular epidermal layer as the scoring region in skin, or the squamous epithelial compartment in tonsil, before reviewing stain intensity (UniProt P20930; HPA tissue IHC; standard IHC practice). Record the percentage of positive cells and, when intensity matters, an H-score calculated from the percentage at each intensity level (standard IHC practice). Normalise positive counts to the number of evaluable cells in that compartment, or report stained area relative to its evaluable area, and document exclusions for damaged tissue (standard IHC practice). Compare sections processed with the same EDTA pH 8.0 retrieval and detection settings, since technical variation can change chromogenic intensity (datasheet PB9870; standard IHC practice).
How can I distinguish true FLG staining from an artefact?
A persuasive positive result follows superficial squamous epithelial cells and has a cytoplasmic distribution; skin granular-layer staining is reported as high and tonsillar squamous epithelial staining as medium (HPA tissue IHC). Check whether a suspected signal instead follows tissue edges, necrotic regions or every cell type, and compare those areas with a no-primary section (standard IHC practice). Nuclear-only signal conflicts with the reported keratohyalin-granule localisation, while residual endogenous peroxidase can produce misleading DAB staining (UniProt P20930; standard IHC practice). Interpret weak or absent staining alongside tissue preservation and the documented EDTA pH 8.0 retrieval, without treating a single negative section as proof of absent FLG (datasheet PB9870; standard IHC practice).
Boster reagents
Best FLG / Filaggrin IHC Antibodies
One human-reactive anti-FLG antibody has real IHC data from a paraffin-embedded human tonsil section (catalog: PB9870; image caption: PB9870).
Real IHC data
Anti-Filaggrin/FLG Antibody ®
Cat # PB9870
PB9870 is listed for human IHC (catalog: PB9870 applications and reactivity). Its IHC figure shows staining in a paraffin-embedded human tonsil section; the fixative is unreported (image caption: PB9870).
Which to pick: Choose PB9870 for human paraffin-section IHC: its caption documents staining at 1 μg/ml after EDTA pH 8.0 retrieval, but does not report the fixative (image caption: PB9870). No IF/ICC or cross-species choice is supported because PB9870 lists only human reactivity and IHC/WB applications; its clonality is unreported (catalog: PB9870).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P20930 (FILA_HUMAN, Filaggrin).
- Human Protein Atlas. FLG tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FLG subcellular location (ICC-IF): Localized to the cytoplasmic bodies..
- Human Protein Atlas. FLG antibody validation summary (4 antibodies).
- Anti-inflammatory effects of amarogentin on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like mice and in HaCat cells. Animal models and experimental medicine 2023 — PMC10272903.
- Calcipotriol/Betamethasone Dipropionate Foam Inhibits Th17 Cytokine Secretion and Improves Epidermal Barrier Markers in a Human Th17 Skin Inflammation Model. Dermatology and therapy 2021 — PMC7859006.
- The Effect and Mechanism of Burnet Gels on Steroid-Dependent Dermatitis in Guinea Pig Model. BioMed research international 2022 — PMC9489357.
- Yes-associated protein regulates autophagy to restore skin barrier function in atopic dermatitis. Frontiers in immunology 2025 — PMC12672505.
- PubMed PMID:16710414 — UniProt-cited evidence.
- PubMed PMID:1429717 — UniProt-cited evidence.
- PubMed PMID:2740331 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FLG (Filaggrin). Expected localisation Superficial squamous-cell cytoplasm (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9870). Validated IHC antibody: PB9870 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Cells in granular layer); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet PB9870); 2. Fixation: Image fixative and duration unreported (datasheet PB9870); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet PB9870); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9870); 8. Primary antibody: Rabbit anti-FLG, 0.5-1μg/ml (datasheet PB9870); 9. Primary incubation: Overnight at 4 °C (datasheet PB9870). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Signal depends on the proportion of granular cells (HPA tissue IHC); Isoform / epitope: No isoforms annotated; one full-length chain (UniProt); Regulation: Granular-layer enrichment (HPA tissue IHC). Evidence: UniProt P20930, HPA tissue IHC · Enhanced, HPA ICC-IF · cytoplasmic bodies., Datasheet PB9870, PMC10272903, PMC7859006. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FLG/immunohistochemistry-ihc-guide.