This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
FLOT1 / Flotillin-1 · IHC design guide
Design Immunohistochemistry for FLOT1
Plan FLOT1 staining in paraffin sections using 2–5 μg/mL catalog antibody (datasheet PA2033). Assess cytoplasmic and membranous staining, with breast glandular cells as a reported high expression reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FLOT1 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 and membranous staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cells in most tissues show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PA2033) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Oral mucosa |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Oral squamous cells may show no staining (HPA tissue IHC) | |
| Regulation | Regulation not specified (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended FLOT1 IHC & IF Protocols
The catalog antibody’s IHC protocol is paired here with published FLOT1 staining methods for liver and ovarian paraffin sections (datasheet PA2033; PMC3694104; PMC12070024).
| Sample | Paraffin-embedded human stomach camcer tissue; fixative not specified (datasheet PA2033) |
| Fixation | Image fixative and duration unreported (datasheet PA2033); 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 PA2033); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PA2033) |
| Primary antibody | Rabbit anti-FLOT1, 2-5 μg/ml (datasheet PA2033) |
| Primary incubation | Overnight at 4 °C (datasheet PA2033) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PA2033) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FLOT1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet PA2033); neither included article specifies a retrieval method (PMC3694104; PMC12070024).
Section 2
What Is the Expected FLOT1 Staining Pattern?
Expect membranous and cytoplasmic FLOT1 staining across many tissues, including high staining in breast glandular cells and cerebral cortical neurons (HPA tissue IHC: Enhanced; medium consistency with RNA). FLOT1 associates with cell membranes, caveolae and endosomes without a transmembrane segment (UniProt O75955 localization and topology).
| Distinct membrane-associated and cytoplasmic chromogen in breast glandular cells or cerebral cortical neurons. | This fits the reported compartments and high staining in those cell types (HPA tissue IHC). Compare staining within intact cells; diffuse color over the whole section is less convincing than a cell-resolved pattern. |
| Strong nuclear staining dominates while the membrane and cytoplasm show little or no signal. | Nuclear localization is absent from the supplied localization records (UniProt O75955; HPA tissue IHC). Treat a predominantly nuclear result as suspect and check controls, detection background and antibody specificity before scoring it as FLOT1. |
| Strong signal appears in oral mucosal squamous epithelium, with the expected glandular or neuronal pattern absent. | HPA reports FLOT1 as not detected in oral mucosal squamous cells and high in the listed glandular cells and neurons (HPA tissue IHC). Investigate cross-reactivity or endogenous detection activity; a single discrepant section cannot establish the cause. |
| Pale, diffuse chromogen covers cells and surrounding tissue without clear cell boundaries. | This does not resolve the reported cytoplasmic and membranous pattern (HPA tissue IHC). Review background in the negative control and optimize blocking, antibody concentration and chromogen development using general IHC practice. |
| No staining appears in a well-preserved breast glandular-cell or cerebral cortical neuronal region. | Those cells are reported as high staining (HPA tissue IHC). Check whether the positive control worked, then review retrieval, antibody incubation and detection as general IHC troubleshooting steps; absence alone does not identify which step failed. |
💡Expected FLOT1 appearanceCall positive a cell-resolved high membranous and cytoplasmic signal in an HPA high-staining cell population, such as breast glandular cells; diffuse field-wide color or dominant nuclear staining is suspect (HPA tissue IHC; UniProt O75955 localization).
| Compartment and topology | FLOT1 is associated with cell membranes, caveolae and endosomes and has no transmembrane segment (UniProt O75955). In paraffin IHC, assess membrane-associated and cytoplasmic staining together; this topology alone does not predict an antibody's epitope accessibility. |
| Choice of positive and comparison tissue | HPA reports high staining in breast glandular cells, cerebral cortical neurons and several other listed populations, but no detection in oral mucosal squamous cells (HPA tissue IHC). Use the latter as a pattern comparison, not proof of absolute biological absence. |
| Breadth and reliability of tissue staining | The tissue profile describes cytoplasmic and membranous expression in most tissues, with low RNA tissue specificity (HPA tissue IHC). Its Enhanced reliability carries a stated caveat: antibody staining and RNA expression have medium consistency (HPA tissue IHC). |
| Antibody validation | Both listed antibodies, HPA001393 and CAB007766, have Enhanced IHC validation (HPA antibodies). That supports comparison with the reported IHC pattern; it does not make every positive cell or staining compartment specific without suitable section controls. |
| Isoforms and modified residues | UniProt lists 2 isoforms and annotated phosphorylation sites, including residues 19, 163, 385 and 387 (UniProt O75955). Antibody epitope data are absent from this payload, so no isoform-specific staining or retrieval effect can be predicted. |
| IF/ICC Q&A | Q: Should IF/ICC look identical to paraffin IHC? A: HPA places IF signal mainly at the Golgi apparatus, marked uncertain, with supported plasma-membrane localization and uncertain vesicles (HPA subcellular). Interpret that separate assay on its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected positive tissue is blank. | A failed retrieval, primary-antibody incubation or detection step is possible; HPA reports high staining in breast glandular cells (HPA tissue IHC). | Check a known-positive section in the same run. Review retrieval and reagent steps against the antibody's validated IHC instructions; the supplied sources give no FLOT1-specific retrieval condition. |
| Every section shows diffuse brown haze. | Nonspecific binding or overdeveloped chromogen can create diffuse IHC background (general IHC practice). | Inspect the negative control, then adjust blocking, antibody concentration and development time according to the validated assay. Score only staining with recognizable cell boundaries and compartments. |
| Chromogen appears in the negative control. | Endogenous enzyme activity or the detection reagents may contribute signal in chromogenic IHC (general IHC practice). | Confirm the primary-antibody omission control and apply the detection system's appropriate endogenous-activity block. Reassess the tissue pattern only after the control background is resolved. |
| Nuclear staining dominates. | The supplied records describe membrane and cytoplasmic or endosomal localization, not nuclear localization (HPA tissue IHC; UniProt O75955). | Compare with a positive control and negative control, examine whether cytoplasmic and membranous staining is also present, and investigate antibody or detection background before assigning a FLOT1 score. |
| Oral mucosal squamous cells stain strongly. | HPA reports FLOT1 as not detected in that cell population (HPA tissue IHC); cross-reactivity or background is possible, but the observation alone cannot distinguish them. | Review tissue identity and control slides, then compare intensity and compartment with an HPA high-staining population processed in the same run (HPA tissue IHC). |
| IF/ICC shows a Golgi-rich pattern that differs from the paraffin IHC section. | HPA's main Golgi IF location is marked uncertain; plasma-membrane IF localization is supported, while tissue IHC is cytoplasmic and membranous (HPA subcellular; HPA tissue IHC). | Interpret each assay against its own HPA record and controls. Do not treat the uncertain IF Golgi call as a required paraffin IHC pattern. |
Experiment evidence
IHC & IF images showing FLOT1 staining
PA2033 · IHCIHC analysis of FLOT1 using anti-FLOT1 antibody (PA2033). FLOT1 was detected in a paraffin-embedded section of human stomach camcer tissue.
PA2033 · IHCIHC analysis of FLOT1 using anti-FLOT1 antibody (PA2033). FLOT1 was detected in a paraffin-embedded section of mouse brain tissue. Heat medi
M05980 · IHCIHC analysis of Flotillin 1 using anti-Flotillin 1 antibody (M05980). Flotillin 1 was detected in a paraffin-embedded section of human breas
M05980 · IHCIHC analysis of Flotillin 1 using anti-Flotillin 1 antibody (M05980). Flotillin 1 was detected in a paraffin-embedded section of human liver
M04517-1 · IHCHumanhepatocellularcarcinoma was stained with anti-Flotillin-1 rabbit antibody
M04517-1 · IHCHumancolon was stained with anti-Flotillin-1 rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137312-FLOT1/tissue/cerebral+cortex#imid_2408544
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137312-FLOT1/tissue/colon#imid_2408486
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137312-FLOT1/tissue/liver#imid_2408471
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000137312-FLOT1/tissue/pancreas#imid_2408430
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001393; green = FLOT1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001393; green = FLOT1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FLOT1 IHC & IF
🧪Run bone marrow first and score staining in hematopoietic cells (HPA: bone marrow hematopoietic cells High). Use oral mucosa as the negative tissue, scoring squamous epithelial cells (HPA: oral mucosa squamous epithelial cells Not detected); any unstained cells on the marrow slide should show counterstain and background only, but the supplied HPA rows do not establish a FLOT1-negative marrow cell type (HPA: bone marrow row).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Oral mucosa (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FLOT1 in A-431, U-251MG, U2OS, Sperm, with annotated localisation: Golgi apparatus (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody where its isotype is known (PA2033 tissue-IHC caption: rabbit primary; standard IHC practice). Process FLOT1 knockout material alongside the test material as a biological specificity control (standard IHC practice).
Block endogenous peroxidase and inspect marrow for background chromogen before scoring (standard IHC practice: hematopoietic tissue and HRP/DAB detection).
⚠️Feasibility: A FLOT1-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected PA2033 paraffin-section caption does not report its fixative (PA2033 tissue-IHC caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required (PA2033 tissue-IHC caption). Whether frozen sections or IF are easier is unreported; marrow endogenous peroxidase can complicate chromogenic scoring (standard IHC practice).
HPA tissue IHC evidence for FLOT1
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FLOT1 IHC Tips
Troubleshoot chromogenic FLOT1 staining in paraffin sections using the PA2033 tissue image and the reported tissue and subcellular patterns (PA2033 caption; HPA tissue IHC; HPA subcellular).
What retrieval should I try first when FLOT1 staining is weak?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet PA2033). The selected PA2033 tissue image used that buffer, followed by 10% goat serum blocking and 2 μg/mL primary antibody overnight at 4°C (PA2033 caption). If staining remains weak, compare a shorter and longer heating interval on adjacent sections while holding antibody concentration, detection and development constant (standard IHC practice). Assess whether membranous and cytoplasmic staining becomes clearer without a parallel rise in diffuse background (HPA tissue IHC; standard IHC practice). Record the retrieval interval alongside the positive control, because the caption does not specify a heating time (PA2033 caption).
Could fixation explain variable FLOT1 staining across my sections?
Target-specific fixation sensitivity is unknown: the selected paraffin tissue caption does not report a fixative (PA2033 caption). Record the actual fixative, fixation duration and processing history for each specimen, then compare sections processed alike before attributing intensity differences to FLOT1 biology (standard IHC practice). If fixation varies, run a small retrieval comparison using EDTA pH 8.0 and the same antibody and detection settings across matched controls (datasheet PA2033; standard IHC practice). Do not infer a preferred fixative or a fixation-dependent epitope effect from tissue staining patterns, protein topology or phosphorylation sites (HPA tissue IHC; UniProt O75955; PA2033 caption).
Should I expect FLOT1 at the membrane or in the cytoplasm?
Score both membranous and cytoplasmic staining in paraffin sections; that combination is reported across most tissues (HPA tissue IHC). FLOT1 is associated with the cell membrane, endosomes and membrane rafts, and has no annotated transmembrane segment (UniProt O75955). A sharply exclusive nuclear pattern would therefore warrant review of the counterstain, nonspecific signal and control sections before biological interpretation (UniProt O75955; standard IHC practice). Compare cells within the same tissue compartment and annotate membrane accentuation separately from granular cytoplasmic signal (HPA tissue IHC; standard IHC practice). HPA also reports Golgi, vesicular and plasma membrane localisation in cell imaging, with differing confidence for those locations (HPA subcellular).
Can this antibody distinguish FLOT1 isoforms or phosphorylation states in IHC?
Do not assign an isoform or phosphorylation state from this chromogenic result alone: FLOT1 has 2 annotated isoforms and several phosphorylated residues, but the supplied caption gives no epitope mapping (UniProt O75955; PA2033 caption). The annotated protein lacks a transmembrane segment, which does not establish where this antibody binds or how retrieval exposes its epitope (UniProt O75955; PA2033 caption). Check the antibody's documented immunogen or epitope before claiming isoform selectivity, then use an independently validated assay if that distinction drives the conclusion (standard IHC practice). Keep retrieval and staining conditions matched across specimens so technical differences do not masquerade as isoform-dependent expression (standard IHC practice).
How should I investigate FLOT1 localisation by IF alongside this IHC guide?
For a separate IF experiment, multiplex FLOT1 with a marker for the expected cell population and include single-stain controls to check channel bleed-through (standard IF practice). Choose fluorophores and filters after inspecting unstained tissue autofluorescence, favoring a spectrally separated channel where background is lowest (standard IF practice). FLOT1 has no annotated transmembrane segment and is reported at membranes and endosomes; permeabilisation should therefore be chosen for the antibody's documented epitope side, which is not supplied here (UniProt O75955; PA2033 caption). Compare gentle permeabilisation against a matched untreated section, then verify that observed membrane and vesicular patterns agree with controls (HPA subcellular; standard IF practice).
How can I reduce diffuse or granular DAB background?
First compare a no-primary section with the stained section to identify signal from detection reagents or endogenous peroxidase (standard IHC practice). The selected PA2033 image used a peroxidase-conjugated secondary and DAB, so include a peroxidase block as a general chromogenic workflow step (PA2033 caption; standard IHC practice). Its reported conditions include 10% goat serum blocking, 2 μg/mL primary overnight at 4°C and 30 minutes of secondary incubation at 37°C (PA2033 caption). If background persists, adjust blocking, washing and DAB development one variable at a time while preserving a comparable positive control (standard IHC practice).
How should I score FLOT1 staining across paraffin specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and eligible cells before scoring, because FLOT1 staining can be both membranous and cytoplasmic (HPA tissue IHC; standard IHC practice). Record the percentage of positive cells and an intensity-based H-score, with membrane and cytoplasm scored separately when their patterns differ (standard IHC practice). Normalize each score to the number of eligible cells or sampled tissue area, and use positive-cell density per mm² when cell counts per area answer the research question (standard IHC practice). Apply the same retrieval, antibody, DAB development and image threshold settings to all compared sections, and exclude folds and necrotic regions by a prespecified rule (standard IHC practice).
How do I distinguish convincing FLOT1 staining from artefact?
Convincing staining should follow interpretable cell boundaries or cytoplasmic structures in viable tissue, consistent with reported membranous and cytoplasmic expression (HPA tissue IHC; standard IHC practice). Examine the counterstain and serial morphology before treating nuclear-only deposits, section-edge intensity or staining concentrated in necrosis as biological signal (UniProt O75955 localisation; standard IHC practice). Compare a no-primary control and the peroxidase-blocked section to identify endogenous enzyme activity or DAB deposition unrelated to primary-antibody binding (standard IHC practice). Use the reported low or absent staining in oral mucosa squamous epithelial cells only as contextual evidence, since HPA rates tissue staining as Enhanced with medium RNA agreement (HPA tissue IHC).
Boster reagents
Best FLOT1 / Flotillin-1 IHC Antibodies
Anti-FLOT1 antibodies have IHC images from human stomach, breast, liver and colon samples, plus mouse and rat brain; two also list IF (catalog image captions and applications).
Real IHC data
Anti-Flotillin 1/FLOT1 Antibody ®
Cat # PA2033
Real IHC data
Anti-Flotillin 1 FLOT1 Rabbit Monoclonal Antibody
Cat # M05980
Real IHC data
Anti-Flotillin-1 Rabbit Monoclonal Antibody
Cat # M04517-1
PA2033 has paraffin-section IHC images from human stomach cancer and mouse and rat brain, and lists IF; M05980 has paraffin-section IHC images from human breast, liver and lung cancers (catalog image captions and applications). M04517-1 has IHC images from human hepatocellular carcinoma and colon and lists IF (catalog image captions and applications).
Which to pick: For paraffin-section IHC, choose PA2033 for the documented 2 μg/ml staining in human stomach cancer or mouse and rat brain, or M05980 for its documented 1:50 staining in human breast, liver and lung cancers; the fixative is unreported in both captions (catalog image captions). For IF, consider PA2033 at its listed 5 μg/ml or the monoclonal M04517-1 at 1:200–1:1000; neither has an IF image here, and ICC validation is unreported (catalog applications, dilutions and image captions). All three list human, mouse and rat reactivity, but PA2033 supplies IHC images for each species (catalog reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75955 (FLOT1_HUMAN, Flotillin-1).
- Human Protein Atlas. FLOT1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. FLOT1 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to the plasma membrane and vesicles..
- Human Protein Atlas. FLOT1 antibody validation summary (2 antibodies).
- High Expression of FLOT1 Is Associated with Progression and Poor Prognosis in Hepatocellular Carcinoma. PloS one 2013 — PMC3694104.
- Upregulation of flotillin-1 promotes invasion and metastasis by activating TGF-β signaling in nasopharyngeal carcinoma. Oncotarget 2016 — PMC4826203.
- FLOT1 Is a Novel Serum Biomarker of Ovarian Cancer Targeted by N6-methyladenosine Modification Inhibition. Cell biology international 2025 — PMC12070024.
- Flotillin1 promotes EMT of human small cell lung cancer via TGF-β signaling pathway. Cancer biology & medicine 2018 — PMC6372910.
- PubMed PMID:11167132 — UniProt-cited evidence.
- PubMed PMID:11042152 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FLOT1 (Flotillin-1). Expected localisation Cytoplasmic and membranous staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PA2033). Validated IHC antibody: PA2033 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Oral mucosa. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human stomach camcer tissue; fixative not specified (datasheet PA2033); 2. Fixation: Image fixative and duration unreported (datasheet PA2033); 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 PA2033); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PA2033); 8. Primary antibody: Rabbit anti-FLOT1, 2-5 μg/ml (datasheet PA2033); 9. Primary incubation: Overnight at 4 °C (datasheet PA2033). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Oral squamous cells may show no staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unspecified (UniProt); Regulation: Regulation not specified (UniProt). Evidence: UniProt O75955, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the Golgi apparatus. In addition localized to…, Datasheet PA2033, PMC3694104, PMC12070024. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FLOT1/immunohistochemistry-ihc-guide.