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FLOT2 / Flotillin-2 · IHC design guide
Design Immunohistochemistry for FLOT2
This guide covers paraffin-section FLOT2 IHC using an IHC-validated catalog antibody (datasheet: PB9623). It helps select controls and score the cytoplasmic and membranous tissue pattern reported by HPA (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FLOT2 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Broad cytoplasmic and membranous staining; endocrine cells high (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9623) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB9623); verify before use. | |
| Caveat | Squamous epithelium and muscle may stain weakly (HPA tissue IHC) | |
| Regulation | No staining regulator established (UniProt) | |
| Isoform / epitope | No isoforms or transmembrane segment annotated (UniProt) |
Section 1
Recommended FLOT2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (PB9623 datasheet) with two published FLOT2 IHC protocols (PMC4496397; PMC6365903).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet PB9623) |
| Fixation | Image fixative and duration unreported (datasheet PB9623); 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 PB9623); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9623) |
| Primary antibody | Rabbit anti-FLOT2, 0.5-1μg/ml (datasheet PB9623) |
| Primary incubation | Overnight at 4 °C (datasheet PB9623) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9623) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FLOT2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic and membranous expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (PB9623 datasheet). The citrate protocol uses another primary antibody (PMC6365903); verify secondary compatibility before adopting it.
Section 2
What Is the Expected FLOT2 Staining Pattern?
FLOT2 should show cytoplasmic and membranous staining across many tissues, with stronger staining in selected cell populations (HPA tissue IHC: ubiquitous expression; Supported reliability, medium consistency with RNA). Its association with the cell membrane, caveolae and endosomes supports a peripheral or vesicular pattern; it has no annotated transmembrane segment (UniProt Q14254). In skin, assess epidermal layers separately because basal and suprabasal cells differ (UniProt Q14254 tissue specificity).
| Membranous staining with some cytoplasmic staining in expected cells. | This fits the reported tissue pattern (HPA tissue IHC). Membrane-associated FLOT2 and its endosomal location make peripheral and intracellular signal plausible (UniProt Q14254). Judge distribution within identified cells, alongside the intensity of the control tissue; HPA reports medium consistency between staining and RNA (HPA tissue IHC: Supported). |
| Strong nuclear-only staining, with little membrane or cytoplasmic signal. | A dominant nuclear pattern conflicts with the reported tissue and subcellular distributions (HPA tissue IHC; HPA ICC-IF: plasma membrane and vesicles; UniProt Q14254). Suspect nonspecific staining or a detection artefact; assess a known-positive tissue and a no-primary control before treating it as FLOT2 (general IHC practice). |
| Strong staining in basal epidermal cells, without the expected suprabasal pattern. | This reverses the reported skin distribution: FLOT2 is described in epidermal layers except the basal layer, and in suprabasal but not basal hair-follicle cells (UniProt Q14254 tissue specificity). Check cell identification and controls; persistent basal staining may reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse chromogen across cells, stroma or the entire section. | Diffuse deposition obscures the reported cytoplasmic and membranous pattern (HPA tissue IHC). It may arise from nonspecific antibody binding, incomplete blocking or endogenous detection activity (general IHC practice). A no-primary control helps separate detection background from antibody-dependent staining (general IHC practice). |
| No detectable staining in a selected high-expression cell population. | An absent signal in glandular endometrial cells or neuronal cerebral-cortex cells conflicts with their reported High staining (HPA tissue IHC: Endometrium, glandular cells; Cerebral cortex, neuronal cells). Verify that the intended cells are present, then review the IHC workflow and controls; one negative section does not establish absence of FLOT2 (general IHC practice). |
💡Expected FLOT2 appearanceCall a section positive when identified cells show discernible cytoplasmic and membranous staining, with strong signal in an HPA High population such as endometrial glandular cells; diffuse section-wide chromogen or dominant nuclear-only staining is suspect (HPA tissue IHC: High in endometrial glandular cells; HPA ICC-IF: plasma membrane and vesicles; general IHC practice).
| Cell population and tissue selection | HPA reports High staining in appendix and colon endocrine cells, endometrial and fallopian-tube glandular cells, and selected brain cells; it lists low staining in several muscle and squamous epithelial populations (HPA tissue IHC). Compare like cell populations, since whole-tissue averages can hide a positive minority (general IHC practice). |
| Skin layer | Epidermal and appendage staining is expected, while dermis and basal epidermal or hair-follicle layers are not described as expressing FLOT2 (UniProt Q14254 tissue specificity). Identify the layer before calling unexpected staining positive or negative (general IHC practice). |
| Compartment and topology | FLOT2 is membrane associated, including caveolae, and is also reported at endosomes; no transmembrane segment is annotated (UniProt Q14254). HPA tissue IHC describes cytoplasmic and membranous staining. Interpret both compartments, without requiring a continuous surface outline in every cell (HPA tissue IHC; general IHC practice). |
| Antibody evidence | The tissue profile has Supported reliability with medium staining–RNA consistency (HPA tissue IHC). HPA lists IHC as Supported for HPA001396 and CAB004395; those entries do not establish that every antibody reproduces the same pattern (HPA antibodies; general IHC practice). |
| IF/ICC Q: What should fluorescence show? | A: Mainly plasma-membrane signal with additional vesicular signal (HPA ICC-IF: both supported). ICC-IF is a separate application; use its own guide for assay setup. The tissue IHC profile describes broader cytoplasmic and membranous staining in paraffin sections (HPA tissue IHC). |
| Chromogenic detection background | Endogenous enzyme activity or nonspecific detection can add chromogen independently of the primary antibody (general IHC practice). Control this possibility before assigning diffuse or unexpected cell staining to FLOT2; HPA tissue staining levels do not identify the source of background in a new section (HPA tissue IHC; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected High cells appear negative. | The intended cell population may be absent, or the IHC detection workflow may have failed (general IHC practice). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Staining is predominantly nuclear. | The distribution conflicts with membrane, vesicular and cytoplasmic evidence (HPA ICC-IF; HPA tissue IHC; UniProt Q14254). | Inspect morphology and compare a known-positive section with a no-primary control. If nuclear signal persists without the expected pattern, treat target assignment as uncertain (general IHC practice). |
| Basal epidermis stains more strongly than suprabasal epidermis. | The apparent layer distribution conflicts with the reported skin pattern (UniProt Q14254 tissue specificity). | Recheck layer identification and staining controls. Investigate cross-reactivity or endogenous detection signal before interpreting basal staining as FLOT2 (general IHC practice). |
| Chromogen covers most of the section diffusely. | Nonspecific binding or endogenous detection activity can obscure cellular localisation (general IHC practice). | Compare with a no-primary control; review blocking, washing and detection conditions, then reassess whether membrane and cytoplasmic signal remains cell associated (general IHC practice; HPA tissue IHC: expected pattern). |
| A low-staining population shows intense, uniform signal. | The result differs from the reported low staining in, for example, skeletal-muscle myocytes or esophageal squamous epithelial cells (HPA tissue IHC). | Verify cell identity and compare a High reference population on the same run. Use detection controls to assess unexpected signal; HPA tissue profiles describe observed patterns, not a universal negative cutoff (HPA tissue IHC; general IHC practice). |
| The membrane outline is weak but intracellular staining is visible. | Cytoplasmic tissue staining and vesicular ICC-IF signal are documented, so a weak outline alone does not exclude FLOT2 (HPA tissue IHC; HPA ICC-IF). | Judge the cellular pattern against an identified High population and controls. Record membrane and cytoplasmic components separately rather than scoring the outline alone (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing FLOT2 staining
PB9623 · IHCIHC analysis of Flotillin 2 using anti-Flotillin 2 antibody (PB9623). Flotillin 2 was detected in a paraffin-embedded section of human mamma
PB9623 · IHCIHC analysis of Flotillin 2 using anti-Flotillin 2 antibody (PB9623). Flotillin 2 was detected in a paraffin-embedded section of mouse intes
PB9623 · IFIF analysis of Flotillin 2 using anti-Flotillin 2 antibody (PB9623). Flotillin 2 was detected in immunocytochemical section of A431 cells. E
M06107-2 · IHCIHC analysis of Flotillin 2 using anti-Flotillin 2 antibody (M06107-2). Flotillin 2 was detected in a paraffin-embedded section of human pla
M06107-2 · IFIF analysis of FLOT2 using anti-FLOT2 antibody (M06107-2). FLOT2 was detected in an immunocytochemical section of SiHa cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132589-FLOT2/tissue/cerebral+cortex#imid_574408
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132589-FLOT2/tissue/colon#imid_574334
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132589-FLOT2/tissue/liver#imid_574327
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132589-FLOT2/tissue/kidney#imid_574328
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA071038; green = FLOT2, blue = nucleus, red = microtubules.
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA071038; green = FLOT2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FLOT2 IHC & IF
🧪Run appendix first and confirm staining in its endocrine cells (HPA: High in appendix endocrine cells). HPA detects FLOT2 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls to establish background, and treat adjacent non-endocrine cells as internal background comparators only if they remain unstained, not as validated FLOT2-negative cells (HPA: no negative tissue rows).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: None in HPA: FLOT2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FLOT2 in RT-4, SH-SY5Y, U2OS, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Run a secondary-only section, a concentration-matched rabbit IgG isotype control, and FLOT2 knockout material if available (selected PB9623 caption: rabbit primary; standard IHC practice). For chromogenic detection, block endogenous peroxidase and assess endogenous biotin background because the reported method uses a biotinylated secondary and streptavidin–biotin detection (selected PB9623 tissue-IHC caption; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative used for the selected paraffin-section image is unreported (selected PB9623 tissue-IHC caption). Heat retrieval in EDTA at pH 8.0 is documented, but a requirement for retrieval is not established; the supplied evidence also does not establish whether frozen sections or IF are easier (selected PB9623 tissue-IHC caption; HPA: ICC-IF localization images). In appendix, check inflammatory leukocytes for endogenous peroxidase background before interpreting chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for FLOT2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: FLOT2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced FLOT2 IHC Tips
Troubleshoot FLOT2 chromogenic IHC in paraffin sections using the catalog antibody’s documented staining conditions and expected localisation.
Which retrieval should I try first for weak FLOT2 staining?
Start with heat-mediated retrieval in EDTA buffer at pH 8.0 (datasheet PB9623). The catalog antibody stained a paraffin section of human mammary cancer tissue after this retrieval, followed by 1 μg/ml primary antibody overnight at 4°C (datasheet PB9623). Keep section thickness, heating time and detection conditions consistent while comparing retrieval runs, and include a section that stained successfully before (standard IHC practice). If staining remains weak, optimise heating time before testing another buffer as a fallback; check tissue preservation and primary-antibody performance alongside any change (standard IHC practice).
Could fixation explain inconsistent FLOT2 staining between paraffin sections?
Target-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative, and its staining result cannot establish one (datasheet PB9623). Record each specimen’s fixative and fixation duration, then compare sections processed with the same EDTA retrieval at pH 8.0 (datasheet PB9623; standard IHC practice). Include a previously staining control section in each run to distinguish a specimen problem from a change in staining conditions (standard IHC practice). If signal differs, review tissue preservation and processing records before changing antibody concentration; do not attribute the difference to a FLOT2-specific fixation effect without direct evidence (standard IHC practice).
Should FLOT2 stain membranes, cytoplasm, or both in tissue?
Assess membrane-associated and vesicular or cytoplasmic staining in the relevant cells: FLOT2 is reported at the cell membrane, caveolae and endosomes (UniProt Q14254), while HPA describes cytoplasmic and membranous tissue staining (HPA tissue IHC). For each section, record membranous and cytoplasmic signal separately rather than combining them into one positive call (standard IHC practice). Compare the pattern with an appropriately processed control stained using EDTA retrieval at pH 8.0 (datasheet PB9623; standard IHC practice). Diffuse signal alone needs scrutiny because HPA’s tissue-staining assessment is Supported with medium consistency against RNA data (HPA tissue IHC).
Can isoforms or epitope accessibility explain a changed FLOT2 pattern?
The supplied record annotates 0 isoforms and no transmembrane segment for FLOT2; it does not identify the catalog antibody’s epitope (UniProt Q14254; datasheet PB9623). Therefore, do not assign a compartment-specific change to an isoform or assume the epitope faces either side of a membrane (UniProt Q14254; standard IHC practice). FLOT2 has an annotated phosphoserine at residue 405, but the payload gives no evidence that this modification changes antibody binding (UniProt Q14254). Compare serial sections under the documented EDTA pH 8.0 retrieval and a consistent detection workflow before investigating epitope accessibility further (datasheet PB9623; standard IHC practice).
How should I assess FLOT2 by IF alongside this IHC result?
Treat IF as a separate assay: the selected antibody evidence documents chromogenic staining of a paraffin section, while HPA reports FLOT2 mainly at the plasma membrane and additionally in vesicles by ICC/IF (datasheet PB9623; HPA subcellular). Multiplex FLOT2 with a validated marker for the expected cell type, and inspect single-marker controls for channel bleed-through (standard IF practice). Choose a fluorophore away from the tissue’s strongest autofluorescence and verify the choice with an unstained section (standard IF practice). Because the antibody epitope’s membrane-facing side is unspecified, establish it before choosing permeabilisation; compare mild permeabilisation with an unpermeabilised control where appropriate (datasheet PB9623; standard IF practice).
What should I check when FLOT2 DAB staining is widespread?
First compare the stained section with no-primary and secondary-only controls to identify detection-system background before changing the FLOT2 interpretation (standard IHC practice). The selected workflow used 10% goat serum blocking, a biotinylated secondary, a streptavidin–biotin complex and DAB (datasheet PB9623). Check peroxidase blocking and, for this biotin-based detection, endogenous biotin controls if staining follows tissue structures unexpectedly (standard IHC practice). Titrate the primary around the documented 1 μg/ml condition while keeping EDTA retrieval at pH 8.0 and development time fixed (datasheet PB9623; standard IHC practice). Widespread signal alone is insufficient evidence of artefact because HPA reports ubiquitous FLOT2 staining (HPA tissue IHC).
How can I quantify FLOT2 IHC without confusing cell abundance with expression? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and cell population before scoring, then report the percentage of positive cells and staining intensity separately (standard IHC practice). For a conventional H-score, multiply the percentage of cells at each intensity grade 0–3 by that grade and sum to obtain 0–300 (standard IHC practice). Score membranous and cytoplasmic staining separately because both patterns are reported for FLOT2 in tissue (HPA tissue IHC). Normalise positive-cell counts to the number of eligible cells, or stained area to analysed tissue area in mm², while excluding folds and necrosis (standard IHC practice). Use identical retrieval, detection and scoring thresholds across compared sections (standard IHC practice).
How do I distinguish credible FLOT2 staining from tissue artefact?
Look for reproducible membranous with possible vesicular or cytoplasmic staining in intact cells, consistent with FLOT2 localisation evidence (UniProt Q14254; HPA tissue IHC; HPA subcellular). In skin, suprabasal epidermis and follicular layers are more plausible than basal layers or dermis according to the supplied tissue-specificity record (UniProt Q14254). Treat staining confined to section edges, folds or necrotic areas cautiously, and check a no-primary control for endogenous enzyme or detection background (standard IHC practice). Compare with the documented EDTA pH 8.0 run conditions and an appropriate control section before calling a changed pattern biological (datasheet PB9623; standard IHC practice).
Boster reagents
Best FLOT2 / Flotillin-2 IHC Antibodies
The catalog includes anti-FLOT2 antibodies with IHC images from human, mouse and rat paraffin sections and IF images from A431 and SiHa cells (catalog image captions).
Real IHC data
Anti-Flotillin 2/FLOT2 Antibody ®
Cat # PB9623
Real IHC data
Anti-Flotillin 2 Antibody ® (monoclonal, 4D8A3)
Cat # M06107-2
PB9623 has paraffin-section IHC images from human mammary cancer, mouse intestine and rat kidney, plus an A431 cell IF image (PB9623 image captions). M06107-2 has a human placenta paraffin-section IHC image and a SiHa cell IF image; its listed reactivity is human, mouse and rat (M06107-2 image captions; catalog reactivity).
Which to pick: For tissue IHC across human, mouse and rat, choose PB9623: its own paraffin-section captions document all three species with EDTA pH 8.0 retrieval (PB9623 IHC image captions). For IF/ICC, M06107-2 offers mouse monoclonal clone 4D8A3 and a SiHa cell IF example; PB9623 also lists IF/ICC and has an A431 cell IF example (catalog host, clone and applications; IF image captions). M06107-2’s tissue IHC caption documents human placenta with EDTA pH 8.0 retrieval; the fixative is unreported in both products’ IHC captions (M06107-2 and PB9623 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14254 (FLOT2_HUMAN, Flotillin-2).
- Human Protein Atlas. FLOT2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. FLOT2 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to vesicles..
- Human Protein Atlas. FLOT2 antibody validation summary (3 antibodies).
- Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β. Oncotarget 2015 — PMC4496397.
- FLOT2 overexpression is associated with the progression and prognosis of human colorectal cancer. Oncology letters 2019 — PMC6365903.
- Insufficient radiofrequency ablation promotes the metastasis of residual hepatocellular carcinoma cells via upregulating flotillin proteins. Journal of cancer research and clinical oncology 2019 — PMC6435628.
- Flotillin-2 is associated with breast cancer progression and poor survival outcomes. Journal of translational medicine 2013 — PMC3765773.
- PubMed PMID:16625196 — UniProt-cited evidence.
- PubMed PMID:8051082 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FLOT2 (Flotillin-2). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9623). Validated IHC antibody: PB9623 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet PB9623); 2. Fixation: Image fixative and duration unreported (datasheet PB9623); 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 PB9623); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9623); 8. Primary antibody: Rabbit anti-FLOT2, 0.5-1μg/ml (datasheet PB9623); 9. Primary incubation: Overnight at 4 °C (datasheet PB9623). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB9623); verify before use.; Caveat: Squamous epithelium and muscle may stain weakly (HPA tissue IHC); Isoform / epitope: No isoforms or transmembrane segment annotated (UniProt); Regulation: No staining regulator established (UniProt). Evidence: UniProt Q14254, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet PB9623, PMC4496397, PMC6365903. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FLOT2/immunohistochemistry-ihc-guide.