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FAT1 / Protocadherin Fat 1 · IHC design guide
Design Immunohistochemistry for FAT1
Plan FAT1 paraffin IHC around variable cytoplasmic staining and distinct gastrointestinal membrane staining (HPA tissue IHC). Duodenum and small-intestine glandular cells show high staining and can serve as positive references (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FAT1 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 | Variable cytoplasmic staining; distinct GI membrane staining (HPA tissue IHC) | |
| Staining pattern | Most tissues show variable cytoplasm; GI tract shows membranes (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A01536-1) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Bone marrow+1 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01536-1) | |
| Caveat | Staining has medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Regulation not specified (UniProt) | |
| Isoform / epitope | No isoforms annotated; extracellular vs cytoplasmic epitope matters (UniProt) |
Section 1
Recommended FAT1 IHC & IF Protocols
The catalog antibody uses Tris-EDTA pH 9.0 heat retrieval (datasheet A01536-1). The published FAT1 IHC option covers coronary and carotid artery sections (PMC5257202 methods).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A01536-1) |
| Fixation | Image fixative and duration unreported (datasheet A01536-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A01536-1); 20 min, 95–100 °C (standard) |
| 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-FAT1, 1:50 recommended; image 1:200 (datasheet A01536-1) |
| 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 | FAT1-positive staining in cells in molecular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues at variable levels. Distinct membranous expression in gastrointestinal tract and high expression in the CNS. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 for the catalog antibody (datasheet A01536-1); the published mouse protocol boils sections in a retrieval solution whose composition is unspecified (PMC5257202 methods).
Section 2
What Is the Expected FAT1 Staining Pattern?
FAT1 is a single-pass membrane protein with a large extracellular region; UniProt also lists a nuclear form (UniProt Q14517 topology and processing). In tissue IHC, expect variable cytoplasmic staining across many tissues, distinct membrane staining in gastrointestinal glandular cells, and strong staining in selected CNS cells (HPA tissue IHC). HPA rates its tissue profile “Enhanced,” while reporting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| A clear cell-edge pattern in duodenal or small-intestinal glandular cells, with strong signal. | This fits the distinct gastrointestinal membrane pattern and High staining reported for these cells (HPA tissue IHC). Assess the stained cells and their location together: FAT1 has a membrane-spanning region and extracellular domain (UniProt Q14517 topology), but HPA also reports cytoplasmic staining in many tissues (HPA tissue IHC). |
| Strong staining in cerebral-cortex or hippocampal neuronal cells, or cerebellar molecular-layer cells. | These are reported High-staining CNS populations (HPA tissue IHC). A positive result there need not look identical to a gastrointestinal membrane pattern: HPA describes high CNS expression alongside variable cytoplasmic staining across tissues (HPA tissue IHC). Compare the observed cell population and compartment with the chosen tissue’s HPA pattern before scoring. |
| Predominantly nuclear staining, or staining confined to a compartment unexpected for the selected tissue. | Treat this as unresolved rather than automatically positive: UniProt lists both cell-membrane and nuclear localization and a nuclear form (UniProt Q14517 localization and processing), whereas HPA’s tissue summary emphasizes cytoplasmic and gastrointestinal membranous patterns (HPA tissue IHC). Check the antibody’s documented epitope and controls before attributing isolated nuclear signal to FAT1 (standard IHC practice). |
| Strong staining in bone-marrow hematopoietic cells or heart-muscle cardiomyocytes. | HPA reports FAT1 as Not detected in these respective cell populations (HPA tissue IHC). Unexpected signal raises concern for cross-reactivity or endogenous chromogenic detection activity; those are possible IHC explanations, not diagnoses from the HPA profile (standard IHC practice). Review the cell identity and a no-primary control before interpreting the stain. |
| Diffuse color across cells, stroma, and slide background, or no signal in a known-positive tissue. | Diffuse deposition lacks the cell-specific pattern needed for interpretation and can reflect nonspecific staining or detection background (standard IHC practice). Conversely, absent signal in High-staining duodenal glandular cells or cerebral-cortex neuronal cells warrants a technical check before calling the specimen negative (HPA tissue IHC; standard IHC practice). |
💡Expected FAT1 appearanceCall an IHC slide positive when staining is cell-associated in the expected population—especially strong glandular-cell membrane staining in duodenum or small intestine, or strong signal in the listed CNS cells (HPA tissue IHC)—while treating uniform background or strong signal in HPA Not-detected cell populations as suspect (HPA tissue IHC; standard IHC practice).
| Membrane topology and antibody epitope | FAT1 has an extracellular region at residues 22–4181, one transmembrane segment at 4182–4202, and a cytoplasmic region at 4203–4588 (UniProt Q14517 topology). The payload gives no epitope for the catalog antibody, so topology alone cannot predict which FAT1 form or compartment it detects. |
| Processing and nuclear interpretation | UniProt lists a signal peptide at residues 1–21, a FAT1 chain at 22–4588, and a nuclear form; it gives no cleavage mechanism here (UniProt Q14517 processing). Nuclear staining therefore needs antibody-specific validation rather than an assumed processing explanation (standard IHC practice). |
| Tissue and cell selection | Duodenal and small-intestinal glandular cells and several listed CNS cell populations are High, while stomach glandular cells are Medium (HPA tissue IHC). Bone-marrow hematopoietic cells and cardiomyocytes are Not detected (HPA tissue IHC). Score the named cell population, since a tissue-wide positive or negative label loses that distinction. |
| Strength of the tissue evidence | The HPA tissue profile is Enhanced but has medium agreement between staining and RNA data (HPA tissue IHC). Its antibody entries list IHC Enhanced for HPA001869 and IHC Approved for HPA023882 (HPA antibodies). These labels support use of the reported patterns as references; they do not establish the performance of another antibody. |
| IF/ICC Q&A: what localization is expected? | HPA summarizes FAT1 subcellular localization as Membrane, but supplies no main-location designation or ICC-IF cell-line images here (HPA subcellular). UniProt also lists nuclear localization (UniProt Q14517 localization). Use the separate IF/ICC guide for that application; this record does not justify an IF-specific staining protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No color in duodenal or small-intestinal glandular cells selected as a positive reference. | These cells are reported High (HPA tissue IHC); absent staining could reflect a failed IHC workflow or antibody performance (standard IHC practice). | Confirm that the expected glandular cells are present, then review the catalog antibody’s IHC-P instructions, detection reagents, and a concurrently stained positive control (standard IHC practice). Do not infer a FAT1-specific fixation effect from this result. |
| CNS reference tissue stains weakly or unevenly. | HPA reports High staining in specified cerebellar, cerebral-cortex, and hippocampal cell populations, not every CNS cell (HPA tissue IHC). | Identify the relevant population and compare staining within that population (HPA tissue IHC). Check section quality, detection performance, and consistency of routine IHC processing across slides (standard IHC practice). |
| Strong color appears in bone-marrow hematopoietic cells or cardiomyocytes. | Both populations are Not detected in the HPA tissue profile (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (standard IHC practice). | Run a no-primary control and review whether the color tracks the expected cell type or persists without primary antibody (standard IHC practice). Reassess the antibody before treating the result as FAT1. |
| Broad haze obscures cell borders and makes the pattern hard to score. | Diffuse background can arise from nonspecific binding or the chromogenic detection system (standard IHC practice). | Check the no-primary control and routine blocking and wash steps; score FAT1 only where signal can be assigned to cells and compartments (standard IHC practice). HPA’s tissue profile does not specify a FAT1-specific remedy (HPA tissue IHC). |
| A slide shows only nuclear staining. | A nuclear FAT1 form is listed, but the supplied record does not identify the antibody epitope or validate this isolated pattern (UniProt Q14517 processing; HPA tissue IHC). | Check the antibody’s epitope documentation and compare a suitable positive tissue and control slides before assigning the nuclear signal to FAT1 (standard IHC practice). Avoid calling it an artifact solely because FAT1 is membrane-associated (UniProt Q14517 localization). |
| Membrane staining is absent while cytoplasmic staining remains visible. | HPA reports variable cytoplasmic staining in most tissues and distinct membranous staining in the gastrointestinal tract (HPA tissue IHC). The expected compartment depends on the examined tissue. | Confirm tissue and cell identity, then compare with that tissue’s HPA pattern (HPA tissue IHC). If gastrointestinal glandular cells are the target, review the catalog antibody’s IHC-P conditions and positive control (standard IHC practice). |
Experiment evidence
IHC & IF images showing FAT1 staining
A01536-1 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000083857-FAT1/tissue/cerebral+cortex#imid_937568
HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000083857-FAT1/tissue/small+intestine#imid_957133
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000083857-FAT1/tissue/colon#imid_957142
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000083857-FAT1/tissue/liver#imid_957159
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FAT1 IHC & IF
🧪Run duodenum first and look for staining in glandular cells (HPA: High in duodenal glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected in bone marrow hematopoietic cells); on the duodenum slide, assess background in morphologically identified unstained cells without assuming a particular cell type is negative (HPA: no negative duodenal cell population specified).
Positive control tissue: Cerebellum (Cells in molecular layer, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for FAT1; derive a cell-line control from the positive tissue's cell type (Cells in molecular layer) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a host-matched nonimmune IgG control; match the isotype for a monoclonal primary, or use host-matched nonimmune IgG for a polyclonal primary (standard IHC practice). Confirm specificity with knockout material or a validated peptide-blocking control if available, and check for endogenous peroxidase signal in duodenal inflammatory cells before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected SKU’s paraffin-section caption does not state a fixative (caption: fixative not stated). The caption uses Tris-EDTA retrieval at pH 9.0 and primary antibody at 1:200, but does not establish whether retrieval is required across specimens (caption: retrieval and dilution). Whether frozen sections or IF/ICC are easier is unreported; assess duodenal inflammatory-cell peroxidase background when scoring chromogenic signal (standard IHC practice).
HPA tissue IHC evidence for FAT1
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 |
|---|---|---|---|---|
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced FAT1 IHC Tips
Troubleshoot FAT1 staining in paraffin section IHC by checking retrieval, staining compartment, tissue context and controls before comparing scores.
What should I adjust if FAT1 staining is weak after antigen retrieval?
Start with heat mediated Tris-EDTA retrieval at pH 9.0 for the catalog antibody (datasheet A01536-1). The documented paraffin section example used that buffer, a 1:200 primary dilution overnight at 4°C, and a 1:200 secondary for 45 minutes at room temperature (datasheet A01536-1). Check heating consistency, section adhesion and cooling before changing several variables together, then compare sections processed in the same run. If staining remains weak, vary heating time cautiously and assess tissue damage alongside signal; the caption does not establish an optimal heating duration (datasheet A01536-1).
Could fixation explain weak or patchy FAT1 staining?
Target specific FAT1 sensitivity to fixation is unknown from the supplied evidence; the catalog image identifies paraffin embedded tonsil but states no fixative (datasheet A01536-1). Record fixative, fixation duration and tissue thickness for each specimen, then compare cases with a consistently processed control section. Excessive or uneven fixation can impair epitope access in routine IHC, while poor preservation can create patchy morphology; neither observation alone establishes a FAT1 specific effect. Keep retrieval at Tris-EDTA pH 9.0 during this comparison (datasheet A01536-1). Judge any change against morphology, background and signal in the same anatomical compartment.
How should I evaluate membranous, cytoplasmic and nuclear FAT1 staining?
Inspect membrane borders and cytoplasm separately: FAT1 has an extracellular region at residues 22–4181 and a cytoplasmic region at 4203–4588 (UniProt Q14517 topology). Cytoplasmic staining occurs in many tissues, while distinct membrane staining is reported in the gastrointestinal tract (HPA tissue IHC profile). UniProt also lists a nuclear location and nuclear form, so nuclear signal needs compartment specific controls rather than automatic rejection (UniProt Q14517 subcellular location and processing). Compare the same compartment across matched sections, checking cell boundaries and counterstained nuclei before assigning a score. Document mixed patterns separately because a single whole tissue intensity can conceal them.
Why might two FAT1 antibodies produce different staining patterns?
First establish each antibody’s immunogen and mapped epitope; none is supplied for the catalog antibody, so its recognized region cannot be assigned (datasheet A01536-1). FAT1 spans the membrane at residues 4182–4202, separating a large extracellular region from a shorter cytoplasmic tail (UniProt Q14517 topology). The extracellular region carries 22 listed glycosylation sites, which makes epitope accessibility a reasonable question when comparing antibodies, though no effect on this antibody is documented (UniProt Q14517 glycosylation). A nuclear form is listed, but no numbered isoforms are provided (UniProt Q14517 processing and isoforms). Compare antibodies on adjacent sections with identical retrieval and score compartments separately.
How can IF help resolve an ambiguous chromogenic FAT1 pattern?
Use IF as a separate validation experiment and multiplex FAT1 with a marker identifying the cell population under study; gastrointestinal glandular cells and neuronal cells are among the reported positive populations (HPA tissue IHC). Choose fluorophores in channels with low measured tissue autofluorescence, and include single stain and no primary controls to check bleed through and background. Before selecting permeabilisation, determine whether the antibody recognizes the extracellular region at 22–4181 or cytoplasmic region at 4203–4588; that epitope is not supplied for the catalog antibody (UniProt Q14517 topology; datasheet A01536-1). Compare IF compartments with the IHC section while allowing for differences in sample preparation and detection.
What controls help distinguish FAT1 signal from IHC background?
Run a no primary control alongside the catalog antibody to reveal signal from the detection system, endogenous enzyme activity or tissue pigment. For peroxidase based chromogenic IHC, use a peroxidase block and inspect the control before treating diffuse DAB deposits as FAT1 staining. The documented example used a 1:200 primary overnight at 4°C and a 1:200 secondary for 45 minutes; those conditions are a comparison point, not proof that every brown deposit is specific (datasheet A01536-1). Check section edges and damaged regions for disproportionate staining. Evaluate retained cell boundaries and the expected compartment against adjacent, well preserved tissue (UniProt Q14517 subcellular location).
How should I score FAT1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports variable cytoplasmic staining and distinct gastrointestinal membrane staining (HPA tissue IHC profile). For a cell based H-score, record the percentage of cells at intensity 0–3 and sum percentage times intensity, giving a 0–300 range. Report membrane and cytoplasmic scores separately, or use percent positive cells when intensity grading is unreliable. Normalise each score to the number of eligible, viable cells in the annotated region and apply the same threshold and imaging conditions across cases. Exclude folds, necrosis and edge staining using rules set before reviewing outcomes.
When is FAT1 staining credible rather than an artefact?
A credible result has intact morphology, a reproducible compartment and a cell population consistent with the tissue context; gastrointestinal glandular cells show reported FAT1 staining (HPA tissue IHC). Interpret cytoplasmic staining with care because it occurs across many tissues, and assess membrane borders separately (HPA tissue IHC profile). Nuclear staining needs independent support because a nuclear FAT1 form is listed but the catalog antibody’s epitope is unspecified (UniProt Q14517 processing; datasheet A01536-1). Treat staining confined to cut edges or necrosis, or reproduced in a no primary control, as suspect. Endogenous enzyme signal should diminish with an appropriate peroxidase block in a peroxidase based assay.
Boster reagents
Best FAT1 / Protocadherin Fat 1 IHC Antibodies
A01536-1 is a human-reactive anti-FAT1 antibody listed for IHC and IF (catalog: applications and reactivity), with a real IHC image of a paraffin-embedded human tonsil section (IHC image caption).
Real IHC data
Anti-Protocadherin Fat 1 FAT1 Antibody
Cat # A01536-1
A01536-1 is the only card and is listed for human IHC and IF (catalog: applications and reactivity). Its image documents IHC on a paraffin-embedded human tonsil section; no IF image is supplied (catalog: image captions).
Which to pick: Choose A01536-1 for human paraffin-section IHC: its own caption documents tonsil staining with Tris-EDTA retrieval at pH 9.0 and primary antibody at 1:200 overnight (IHC image caption). A01536-1 is also listed for IF at 1:50, but the payload supplies no IF image or ICC validation (catalog: applications, IF dilution and image captions). Its listed reactivity is Human, so cross-species use is unvalidated; its host is Rabbit and clonality is unreported (catalog: reactivity, host and clone), and the caption does not report the tissue fixative (IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14517 (FAT1_HUMAN, Protocadherin Fat 1).
- Human Protein Atlas. FAT1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FAT1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. FAT1 antibody validation summary (2 antibodies).
- De novo Membranous Nephropathy in Renal Allografts is Associated With Protocadherin FAT1. Kidney international reports 2025 — PMC11725789.
- A pan-cancer analysis of the FAT1 in human tumors. Scientific reports 2022 — PMC9751142.
- Control of mitochondrial function and cell growth by the atypical cadherin Fat1. Nature 2016 — PMC5257202.
- Protocadherin FAT1-associated membranous nephropathy after hematopoietic stem cell transplantation-a cohort study with clinico-pathological correlations. Clinical kidney journal 2026 — PMC13098137.
- PubMed PMID:8586420 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
- PubMed PMID:15922730 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FAT1 (Protocadherin Fat 1). Expected localisation Variable cytoplasmic staining; distinct GI membrane staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A01536-1). Validated IHC antibody: A01536-1 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in molecular layer); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A01536-1); 2. Fixation: Image fixative and duration unreported (datasheet A01536-1); 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: Tris-EDTA pH 9.0 (datasheet A01536-1); 20 min, 95–100 °C (standard); 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-FAT1, 1:50 recommended; image 1:200 (datasheet A01536-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01536-1); Caveat: Staining has medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms annotated; extracellular vs cytoplasmic epitope matters (UniProt); Regulation: Regulation not specified (UniProt). Evidence: UniProt Q14517, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A01536-1, PMC5257202. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FAT1/immunohistochemistry-ihc-guide.