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FRMD6 / FERM domain-containing protein 6 · IHC design guide
Design Immunohistochemistry for FRMD6
Plan FRMD6 staining in paraffin sections using the catalog antibody at 0.5–1 μg/mL with chromogenic detection (datasheet A08826-2). Expect cytoplasmic staining in several tissue types, while interpreting intensity cautiously because antibody staining and RNA expression show low consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FRMD6 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 tissue staining (HPA tissue IHC); membrane location (UniProt) | |
| Staining pattern | Cytoplasmic staining in several tissue types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08826-2) | |
| Positive control | Bone marrow+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 | Staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | No target-specific regulator annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; assess epitope coverage across variants (UniProt) |
Section 1
Recommended FRMD6 IHC & IF Protocols
The catalog antibody has a paraffin-section IHC protocol (datasheet: A08826-2). Published FRMD6 IHC methods include colorectal tissue, frozen mouse neural tissue, and colon carcinoma tissue microarrays (PMC6382106; PMC7002571; PMC8073668).
| Sample | Paraffin-embedded human rectum cancer tissue; fixative not specified (datasheet A08826-2) |
| Fixation | Image fixative and duration unreported (datasheet A08826-2); 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 A08826-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08826-2) |
| Primary antibody | Rabbit anti-FRMD6, 0.5-1μg/ml (datasheet A08826-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08826-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A08826-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FRMD6-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A08826-2). The published colorectal paraffin protocol used Tris-EDTA pH 9.0 (PMC6382106).
Section 2
What Is the Expected FRMD6 Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic FRMD6 staining in several tissue types (HPA tissue IHC: cytoplasmic expression). Membrane-associated staining is plausible because FRMD6 is listed in the cytoplasm and at the cell membrane, with no transmembrane segment (UniProt Q96NE9: localisation and topology). HPA reports medium staining in selected epithelial, tubular, hepatic and hematopoietic cells, but rates its tissue IHC reliability Uncertain because staining and RNA expression have low consistency (HPA tissue IHC).
| Cytoplasmic chromogen in kidney tubule cells or colon glandular cells. | This fits the reported compartment and two medium-staining cell populations (HPA tissue IHC: kidney and colon). Compare signal within the named cells, rather than treating every cell in the section as an expected positive. The Uncertain tissue IHC reliability limits how confidently this pattern alone identifies FRMD6 (HPA tissue IHC). |
| A membrane outline accompanies cytoplasmic staining. | Membrane association is compatible with the recorded localisation, although HPA describes the tissue IHC pattern as cytoplasmic (UniProt Q96NE9: cell membrane and cytoplasm; HPA tissue IHC). A membrane outline alone is weaker evidence than staining in the expected cell population; no transmembrane segment is annotated (UniProt Q96NE9: topology). |
| Predominantly nuclear staining, with little cytoplasmic signal. | This differs from the supplied FRMD6 localisations (UniProt Q96NE9: cytoplasm and cell membrane; HPA tissue IHC: cytoplasmic expression). Treat it as a possible nonspecific or detection-related pattern. Reassess the control slide and staining distribution before assigning nuclear positivity to FRMD6. |
| Chromogen concentrates in adipocytes or salivary glandular cells. | Those named cells were reported as not detected, so prominent staining warrants scrutiny for nonspecific binding or endogenous detection activity (HPA tissue IHC: adipose tissue and salivary gland). Because tissue IHC reliability is Uncertain, their reported absence is a comparison point, not proof that every specimen must be negative (HPA tissue IHC). |
| Diffuse colour covers cells and surrounding tissue, or a kidney section has no tubular signal. | Diffuse colour obscures the cytoplasmic pattern reported by HPA; inspect the negative detection control for background (HPA tissue IHC: cytoplasmic expression; general IHC practice). For absent tubular staining, check the positive control and staining run before interpreting the specimen: kidney tubule cells had medium staining in the HPA record (HPA tissue IHC: kidney). |
💡Expected FRMD6 appearanceA credible positive is discernible cytoplasmic chromogen in reported medium-staining cells, such as kidney tubule or colon glandular cells; an isolated nuclear or widespread cell-independent deposit is suspect, and the call remains provisional because tissue IHC reliability is Uncertain (HPA tissue IHC; UniProt Q96NE9: localisation).
| Compartment and topology | Use cytoplasmic staining as the tissue IHC reference pattern (HPA tissue IHC). Membrane-associated signal can be considered alongside it, but FRMD6 has no annotated transmembrane segment (UniProt Q96NE9: localisation and topology). |
| Antibody evidence | The listed antibody HPA001297 is rated IHC Uncertain, while the overall tissue IHC profile is Uncertain because staining and RNA expression have low consistency (HPA antibodies; HPA tissue IHC). Give any single staining pattern limited weight. |
| Isoforms and modifications | Three isoforms and several phosphorylated residues are annotated (UniProt Q96NE9: isoforms and modified residues). The supplied record gives no antibody epitope or evidence that these features change IHC staining; do not infer an isoform-specific or phosphorylation-specific pattern. |
| ICC/IF evidence — Q: Should mitochondrial IF dictate the IHC call? | A: No mitochondrial tissue IHC pattern is supplied. HPA reports an approved mitochondrial ICC/IF location, while its tissue IHC description is cytoplasmic and Uncertain (HPA subcellular ICC/IF; HPA tissue IHC). Assess each application against its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining appears in kidney tubule cells. | The staining run may have failed, or signal may be below detection; kidney tubule cells were medium in the HPA tissue record (HPA tissue IHC: kidney). | Check a concurrently stained positive section, antibody omission control, retrieval and detection steps (general IHC practice). Avoid calling the specimen FRMD6-negative until the run works; HPA tissue IHC reliability is Uncertain (HPA tissue IHC). |
| The section stains mainly in nuclei. | The dominant compartment conflicts with cytoplasmic tissue staining and the recorded cytoplasm/cell membrane localisation (HPA tissue IHC; UniProt Q96NE9). | Compare the positive and antibody omission controls, then review whether chromogen truly lies inside nuclei rather than over adjacent cytoplasm (general IHC practice). Treat persistent nuclear-only staining as unverified for FRMD6. |
| Colour appears across cells and cell-free areas. | Diffuse deposition can hide the expected cytoplasmic distribution (HPA tissue IHC: cytoplasmic expression); a detection or wash background problem is possible (general IHC practice). | Inspect the antibody omission control and reagent background; review blocking, washes and chromogen development (general IHC practice). Score cell-associated signal only when its boundaries can be distinguished. |
| Strong staining appears in adipocytes or salivary glandular cells. | These cells are listed as not detected, so unexpected colour may reflect nonspecific binding or endogenous detection activity (HPA tissue IHC: adipose tissue and salivary gland; general IHC practice). | Compare controls and a reported positive cell population on the same run; investigate endogenous activity if the detection chemistry warrants it (general IHC practice). Do not use one reported negative population as an absolute exclusion, given Uncertain tissue IHC reliability (HPA tissue IHC). |
| Only a narrow membrane rim stains in an otherwise negative section. | FRMD6 can associate with the cell membrane, but HPA describes tissue IHC expression as cytoplasmic (UniProt Q96NE9: localisation; HPA tissue IHC). | Review the same cell type for cytoplasmic colour and compare staining with controls (general IHC practice). Record membrane-only staining separately as equivocal until supported by a reproducible pattern; the supplied tissue IHC assessment is Uncertain (HPA tissue IHC). |
| Mitochondrial-looking puncta dominate the tissue IHC slide. | Mitochondria are the approved HPA ICC/IF location, but the supplied tissue IHC description is cytoplasmic and Uncertain (HPA subcellular ICC/IF; HPA tissue IHC). | Assess the IHC slide against tissue cell-type patterns and controls (general IHC practice). Do not transfer the ICC/IF location into an IHC scoring rule without application-specific evidence. |
Experiment evidence
IHC & IF images showing FRMD6 staining
A08826-2 · IHCIHC analysis of FRMD6 using anti-FRMD6 antibody (A08826-2). FRMD6 was detected in paraffin-embedded section of human rectum cancer tissue. H
A08826-2 · IFIF analysis of FRMD6 using anti-FRMD6 antibody (A08826-2). FRMD6 was detected in immunocytochemical section of A431 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000139926-FRMD6/tissue/cerebral+cortex#imid_445450
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000139926-FRMD6/tissue/colon#imid_445376
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000139926-FRMD6/tissue/liver#imid_445369
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000139926-FRMD6/tissue/kidney#imid_445378
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001297; green = FRMD6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001297; green = FRMD6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FRMD6 IHC & IF
🧪Run colon first: glandular cells should stain (HPA: Medium in colon glandular cells). Use adipose tissue as the negative tissue, assessing adipocytes (HPA: Not detected in adipocytes); within the colon slide, cells without specific staining should show counterstain without brown DAB deposits, but the supplied HPA row does not identify a particular internal negative cell type.
Positive control tissue: Bone marrow (Hematopoietic cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FRMD6 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched nonimmune rabbit IgG isotype control (caption: rabbit primary), and FRMD6-knockout material as a biological negative. Quench endogenous peroxidase and assess endogenous biotin background when using the caption’s SABC/DAB detection (caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A08826-2 tissue-IHC caption does not state its fixative. Heat retrieval in EDTA at pH 8.0 is a documented paraffin-section starting condition, but its necessity for colon has not been established by the supplied evidence (caption: EDTA pH 8.0 retrieval in rectum cancer tissue). Is IF/ICC easier? The supplied evidence does not establish that frozen sections or IF are easier; IF localisation should be checked independently because HPA reports mitochondria whereas UniProt annotates cytoplasm and cell membrane (HPA: approved mitochondrial ICC-IF location; UniProt Q96NE9: cytoplasm and cell membrane).
HPA tissue IHC evidence for FRMD6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FRMD6 IHC Tips
Use the validated paraffin-section workflow as the starting point, and interpret FRMD6 staining with compartment and tissue controls (datasheet A08826-2; UniProt Q96NE9; HPA tissue IHC).
Which retrieval conditions should I start with for FRMD6 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08826-2). Keep heating and cooling conditions consistent across slides, then compare staining with a section processed without primary antibody (standard IHC practice). The documented workflow detected FRMD6 in a paraffin section of human rectum cancer using 1 µg/mL primary antibody overnight at 4°C (datasheet A08826-2). If staining remains weak, test another retrieval buffer or heating duration on matched sections as a fallback, while checking tissue preservation and background before adopting the change (standard IHC practice).
How should I troubleshoot FRMD6 staining when fixation varies between specimens?
The selected paraffin-section caption does not report a fixative, so FRMD6-specific sensitivity to fixation is unknown (datasheet A08826-2). Record each specimen’s fixative and fixation time, and compare sections processed with the same retrieval and detection conditions (standard IHC practice). Start those comparisons with EDTA retrieval at pH 8.0 and the documented 1 µg/mL primary antibody concentration (datasheet A08826-2). If signal differs, assess morphology and no-primary controls before attributing the difference to fixation; neither tissue staining patterns nor FRMD6 topology establishes a target-specific fixation effect (standard IHC practice; HPA tissue IHC; UniProt Q96NE9).
Where should FRMD6 staining appear, and how should I handle conflicting compartment patterns?
Assess cytoplasmic and cell-edge staining because FRMD6 is annotated in the cytoplasm and at the cell membrane, with possible actin colocalisation (UniProt Q96NE9). Its lack of a transmembrane segment makes a sharp membrane outline insufficient by itself to establish specificity (UniProt Q96NE9; standard IHC practice). HPA reports cytoplasmic tissue staining but an approved mitochondrial location in cell images, so compare compartment patterns without assuming they must agree (HPA tissue IHC; HPA subcellular). Review matched no-primary controls and preserved cellular morphology, and document diffuse cytoplasmic, peripheral and punctate signal separately before assigning a localisation (standard IHC practice).
Could isoforms or epitope location explain inconsistent FRMD6 staining?
FRMD6 has 3 annotated isoforms and a FERM domain spanning residues 16–328 (UniProt Q96NE9). Before comparing antibodies or specimens, obtain the catalog antibody’s stated immunogen or mapped epitope and check whether it is shared across those isoforms (standard IHC practice). The record also lists phosphorylation near residues 522–544, but it does not establish that these modifications alter this antibody’s staining (UniProt Q96NE9). If epitope coverage is unknown, describe the result as antibody-dependent FRMD6 immunoreactivity; compare matched sections and, where available, an independently validated antibody before making isoform-specific claims (standard IHC practice).
How can IF help resolve ambiguous FRMD6 staining seen by chromogenic IHC?
Use IF as a complementary localisation check on matched material, while keeping the chromogenic paraffin-section workflow as the IHC reference (datasheet A08826-2; standard IHC practice). Multiplex FRMD6 with a validated marker for the expected cell population, such as glandular cells in colon, where HPA reports medium staining (HPA tissue IHC). Choose a fluorophore channel after measuring tissue autofluorescence, and include single-label and no-primary controls to evaluate bleed-through and background (standard IF practice). Because FRMD6 lacks a transmembrane segment and has cytoplasmic localisation, use permeabilisation when testing an intracellular or cytoplasmic-face epitope, subject to the antibody’s stated epitope accessibility (UniProt Q96NE9; standard IF practice).
How do I reduce diffuse or granular background without losing FRMD6 signal?
First compare the stained section with a no-primary control and inspect whether signal tracks tissue edges, folds or damaged regions (standard IHC practice). The documented workflow used 10% goat serum, 1 µg/mL rabbit primary overnight at 4°C, and biotin-based detection with DAB (datasheet A08826-2). Keep those conditions fixed while checking blocking, washing, endogenous peroxidase activity and detection-system background on matched sections (standard IHC practice). If granular staining persists, assess it against preserved cytoplasmic or peripheral cell staining and a second detection control before calling it FRMD6; mitochondrial localisation has also been reported in cell images (UniProt Q96NE9; HPA subcellular; standard IHC practice).
What should I score when comparing FRMD6 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, and use the same threshold, retrieval and DAB development conditions for every comparison (standard IHC practice). For epithelial regions, report the percentage of positive cells and an H-score based on staining intensity; for mixed tissue, density per mm² can describe positive cells (standard IHC practice). Normalise counts to viable, evaluable tissue area or to the number of cells in the predefined population, excluding folds and necrosis (standard IHC practice). Record cytoplasmic and peripheral staining separately because both are plausible FRMD6 locations, and disclose that HPA rates its tissue IHC reliability as uncertain (UniProt Q96NE9; HPA tissue IHC).
When is FRMD6 staining convincing rather than an artefact?
Look for reproducible staining in intact cells with cytoplasmic or peripheral localisation, consistent with FRMD6’s annotated locations (UniProt Q96NE9; standard IHC practice). HPA reports medium staining in colon glandular cells and kidney tubule cells, yet rates overall tissue IHC reliability as uncertain because staining and RNA data show low consistency (HPA tissue IHC). Treat signal confined to section edges, necrosis or a no-primary control as suspect, and check endogenous enzyme or detection background before scoring it (standard IHC practice). A wholly nuclear pattern or staining restricted to an unexpected cell population needs independent validation; compartment and cell identity alone cannot prove specificity (UniProt Q96NE9; HPA tissue IHC; standard IHC practice).
Boster reagents
Best FRMD6 / FERM domain-containing protein 6 IHC Antibodies
A08826-2 has IHC data from human rectum cancer paraffin sections and IF/ICC data from A431 cells (catalog image captions); listed reactivity includes human, mouse and rat (catalog reactivity).
Real IHC data
Anti-FRMD6/Willin Antibody ®
Cat # A08826-2
A08826-2 will render with an IHC image from a paraffin section of human rectum cancer tissue (IHC image caption). The same SKU has IF/ICC data from A431 cells (IF image caption) and lists human, mouse and rat reactivity (catalog reactivity).
Which to pick: Choose A08826-2 for tissue IHC: its IHC caption documents a human rectum cancer paraffin section, EDTA retrieval at pH 8.0, and 1 μg/ml primary antibody; the fixative is unreported (IHC image caption). Choose A08826-2 for IF/ICC: its IF caption documents A431 cells with 2 μg/ml primary antibody (IF image caption). For mouse or rat samples, A08826-2 lists species reactivity, but the supplied IHC and IF images show human samples only (catalog reactivity; IHC and IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96NE9 (FRMD6_HUMAN, FERM domain-containing protein 6).
- Human Protein Atlas. FRMD6 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. FRMD6 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. FRMD6 antibody validation summary (1 antibodies).
- Early-stage serrated adenocarcinomas are divided into several molecularly distinct subtypes. PloS one 2019 — PMC6382106.
- Expression and regulation of FRMD6 in mouse DRG neurons and spinal cord after nerve injury. Scientific reports 2020 — PMC7002571.
- PD-L1 as a Prognostic Factor in Early-Stage Colon Carcinoma within the Immunohistochemical Molecular Subtype Classification. Cancers 2021 — PMC8073668.
- FRMD6 inhibits human glioblastoma growth and progression by negatively regulating activity of receptor tyrosine kinases. Oncotarget 2016 — PMC5342536.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FRMD6 (FERM domain-containing protein 6). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); membrane location (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08826-2). Validated IHC antibody: A08826-2 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human rectum cancer tissue; fixative not specified (datasheet A08826-2); 2. Fixation: Image fixative and duration unreported (datasheet A08826-2); 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 A08826-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08826-2); 8. Primary antibody: Rabbit anti-FRMD6, 0.5-1μg/ml (datasheet A08826-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08826-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; assess epitope coverage across variants (UniProt); Regulation: No target-specific regulator annotated (UniProt). Evidence: UniProt Q96NE9, HPA tissue IHC · Uncertain, HPA ICC-IF · mitochondria., Datasheet A08826-2, PMC6382106, PMC7002571. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FRMD6/immunohistochemistry-ihc-guide.