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REST / RE1-silencing transcription factor · IHC design guide
Design Immunohistochemistry for REST
Plan paraffin-section REST IHC around its general nuclear tissue pattern (HPA tissue IHC) and the catalog antibody’s 2–5 μg/ml IHC range (datasheet A03852-3). Score nuclear staining by cell type, accounting for hypoxia-associated nuclear increases (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
REST 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 | Nuclear tissue staining (HPA tissue IHC); cytoplasm also annotated (UniProt) | |
| Staining pattern | General nuclear staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03852-3) | |
| Positive control | Bronchus+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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03852-3) | |
| Caveat | Neuronal nuclear REST can decrease in dementia (UniProt) | |
| Regulation | Hypoxia increases nuclear REST (UniProt) | |
| Isoform / epitope | Four isoforms; validate antibody coverage of each (UniProt) |
Section 1
Recommended REST IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet A03852-3). Published IHC protocols cover Merkel cell carcinoma (PMC6226622) and brain sections (PMC5797046).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A03852-3) |
| Fixation | Image fixative and duration unreported (datasheet A03852-3); 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 A03852-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03852-3) |
| Primary antibody | Rabbit anti-REST, 2-5 μg/ml (datasheet A03852-3) |
| Primary incubation | Overnight at 4 °C (datasheet A03852-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03852-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | REST-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A03852-3); the brain-section protocol reports heat retrieval without specifying a buffer (PMC5797046).
Section 2
What Is the Expected REST Staining Pattern?
REST is mainly nuclear, with additional cytoplasmic localization (UniProt Q13127; HPA: general nuclear IHC expression; HPA: nucleoplasm and cytosol in ICC-IF). In paraffin sections, look for nuclear staining in respiratory epithelial, squamous epithelial, glandular and alveolar type I cells listed as high by HPA (HPA tissue IHC). REST has no transmembrane segment, so a membrane outline is unexpected (UniProt Q13127 topology). HPA rates its tissue IHC evidence Supported, with medium consistency and external verification pending (HPA tissue IHC).
| Distinct nuclear chromogen in bronchial respiratory epithelium, with nuclei identifiable beneath the counterstain. | This fits a high-staining cell population and REST’s principal nuclear location (HPA: bronchus respiratory epithelial cells, High; HPA: general nuclear IHC expression; UniProt Q13127). Score the stained nuclei within that cell population rather than treating all structures in the section as equivalent. |
| Nuclear staining in endometrial glandular cells; weaker cytoplasmic color is also visible. | The nuclear component fits the expected pattern (HPA: endometrium glandular cells, High; UniProt Q13127: nucleus). Some cytoplasmic REST is plausible (UniProt Q13127: cytoplasm; HPA ICC-IF: additional cytosol). Record the two compartments separately; cytoplasmic color alone does not demonstrate the expected nuclear pattern. |
| A crisp cell-surface rim or extracellular deposit dominates while nuclei lack signal. | A dominant membrane or extracellular pattern is inconsistent with REST’s reported localization and lack of a transmembrane segment (UniProt Q13127; HPA: general nuclear IHC expression). Treat it as suspect staining and review morphology and detection controls before assigning it to REST. |
| Strong signal dominates a population HPA lists as low, such as cerebral cortex glial cells. | This disagrees with the supplied tissue profile (HPA: cerebral cortex glial cells, Low), but is not proof of cross-reactivity: REST is reported as ubiquitous (UniProt Q13127). Check cell identification and nuclear localization; use detection controls to assess endogenous activity before interpreting the difference. |
| The chromogen is diffuse across cells and tissue spaces, or absent in a high-staining reference area. | Diffuse color does not resolve the expected nuclear pattern (HPA: general nuclear IHC expression). Absence from a listed high-staining population, such as bronchial respiratory epithelium, makes the run inconclusive for that specimen (HPA: bronchus, High). Compare controls and section quality before calling biological loss. |
💡Expected REST appearanceCall a positive result when distinct nuclear staining is present in an HPA high-staining cell population, such as bronchial respiratory epithelial cells (HPA: High; HPA: general nuclear IHC expression); additional cytoplasmic signal can occur (UniProt Q13127), while a dominant membrane rim is suspect (UniProt Q13127 topology).
| Cell population and tissue | HPA reports high staining in bronchial respiratory, cervical and esophageal squamous, several glandular, and lung alveolar type I cells, but low staining in specified glial and other populations (HPA tissue IHC). Identify the cells before comparing intensity across sections. |
| Intracellular distribution | REST is reported in nucleus and cytoplasm (UniProt Q13127), while ICC-IF places it mainly in nucleoplasm with additional cytosol (HPA ICC-IF). Nuclear enrichment can increase in hypoxia and aging neurons (UniProt Q13127); interpret a changed nuclear-to-cytoplasmic balance in its specimen context. |
| Isoform coverage | Four REST isoforms are listed (UniProt Q13127). The supplied evidence does not map the IHC antibody’s epitope across them, so a staining difference cannot be assigned to one isoform without additional antibody information. |
| Strength of tissue evidence | HPA labels tissue IHC Supported, describes medium agreement with RNA, and says external verification is pending (HPA tissue IHC). Use its high and low entries as comparison points, not absolute positive or negative guarantees for every specimen. |
| Antigen retrieval and detection | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear stain in bronchial respiratory epithelium that is present and morphologically intact (HPA: High). | The result conflicts with an HPA high-staining reference population; the supplied sources cannot distinguish technical failure from specimen variation (HPA tissue IHC). | Check the run’s positive and detection controls, then review retrieval, primary incubation and chromogen development against the established IHC procedure (general IHC practice). |
| Broad brown color obscures nuclei and fills tissue spaces. | The expected pattern is general nuclear expression (HPA tissue IHC); diffuse color can arise from nonspecific staining or detection background (general IHC practice). | Review the no-primary detection control, blocking, washes and chromogen development; rescore only where individual cells and nuclei can be distinguished (general IHC practice). |
| Strong staining appears mainly at cell borders. | A border-dominant pattern conflicts with nuclear and cytoplasmic REST localization and its lack of a transmembrane segment (UniProt Q13127). | Compare with a listed high-staining tissue area and the detection control; document the border pattern separately instead of counting it as nuclear REST (HPA tissue IHC; general IHC practice). |
| A low-staining HPA population shows stronger signal than nearby expected-positive cells. | The comparison differs from HPA’s cell-specific levels, although ubiquitous REST expression means that an unexpected cell is not automatically negative (HPA tissue IHC; UniProt Q13127). | Recheck cell identity, nuclear localization, background and detection controls; report the discrepancy rather than labeling it cross-reactivity without supporting evidence. |
| Nuclear and cytoplasmic scores differ between specimens. | Both compartments are reported for REST, and nuclear enrichment is described in hypoxia and aging neurons (UniProt Q13127; HPA ICC-IF). The sources do not establish a fixation-based explanation. | Record each compartment and the relevant specimen context separately; compare like cell populations and avoid attributing the shift to retrieval or fixation without further evidence. |
| Can an IF/ICC image be used to judge an IHC section? | HPA ICC-IF supports mainly nucleoplasmic with additional cytosolic localization; tissue IHC has its own Supported assessment and general nuclear profile (HPA ICC-IF; HPA tissue IHC). | Use ICC-IF only as a compartment comparison. Judge paraffin chromogenic staining against tissue IHC cell populations, section morphology and IHC controls (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing REST staining
A03852-3 · IHCIHC analysis of REST using anti-REST antibody (A03852-3). REST was detected in a paraffin-embedded section of human colon cancer tissue. Hea
A03852-3 · IHCIHC analysis of REST using anti-REST antibody (A03852-3). REST was detected in a paraffin-embedded section of human colon tissue. Heat media
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000084093-REST/tissue/cerebral+cortex#imid_18840587
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000084093-REST/tissue/colon#imid_18840513
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000084093-REST/tissue/liver#imid_18840514
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000084093-REST/tissue/kidney#imid_18840499
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006079; green = REST, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006079; green = REST, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for REST IHC & IF
🧪Run bronchus first and assess respiratory epithelial cells for nuclear REST staining (HPA: High in bronchus respiratory epithelial cells; UniProt Q13127: nucleus). HPA detects REST in all 45 scored tissues, so no negative tissue is available; use no-primary and isotype slides for the negative comparison, and expect cells without specific staining on the positive slide to show only background without treating them as a validated biological negative (HPA: no negative rows; standard IHC practice).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: None in HPA: REST 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 REST in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a species- and clonality-matched rabbit IgG control, then confirm specificity with REST knockout material where available (selected tissue-IHC caption: rabbit anti-REST; standard IHC practice). For chromogenic staining of bronchus, quench endogenous peroxidase and compare any residual signal with the control slides (selected tissue-IHC caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A REST-specific fixation window or fixation effect is unreported, and the selected A03852-3 paraffin-section caption does not state the fixative (selected tissue-IHC caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but it does not establish that REST staining depends on that retrieval condition (selected tissue-IHC caption: EDTA retrieval). HPA reports nucleoplasmic and cytosolic ICC-IF signal, but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC; in bronchus, endogenous peroxidase can complicate HRP/DAB interpretation (HPA: subcellular ICC-IF; standard IHC practice).
HPA tissue IHC evidence for REST
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: REST is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced REST IHC Tips
Troubleshoot REST staining in paraffin sections by checking retrieval, nuclear localisation, antibody controls, and cell-specific scoring.
What retrieval should I use when REST staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03852-3). The selected paraffin-section example used this retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C (caption A03852-3). If nuclear staining is weak, check heating consistency and section adhesion before changing retrieval conditions; compare any adjusted condition on matched sections with the same detection exposure (standard IHC practice). Score nuclear signal against background in the same cell population, because REST is mainly nucleoplasmic but can also occur in the cytoplasm (HPA subcellular; UniProt Q13127 localisation).
Could fixation explain absent or uneven REST staining?
The selected image identifies a paraffin-embedded section but does not report its fixative, so target-specific fixation sensitivity is unknown (caption A03852-3). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). Compare matched sections processed with a consistent fixation schedule and the same EDTA pH 8.0 retrieval, primary concentration, and detection conditions (datasheet A03852-3; standard IHC practice). If signal varies across a section, inspect tissue preservation and staining controls before attributing that variation to REST biology (standard IHC practice).
Should REST staining be nuclear, cytoplasmic, or both?
Prioritise nuclear staining: tissue IHC shows general nuclear expression, while cell imaging places REST mainly in the nucleoplasm with additional cytosolic localisation (HPA tissue IHC; HPA subcellular). Cytoplasmic signal is plausible, but score it separately from nuclear signal rather than combining both into one positive call (UniProt Q13127 localisation; standard IHC practice). Hypoxia and aging neurons can show increased nuclear REST relative to cytoplasm, so document the specimen context when comparing compartments (UniProt Q13127, PubMed:27531581, PubMed:24670762, PubMed:30684677). Use a counterstain to define nuclei and review ambiguous cells at higher magnification (standard IHC practice).
Can this stain distinguish REST isoforms or phosphorylation states?
REST has 4 annotated isoforms and modified residues including phosphoserine at positions 864 and 971 (UniProt Q13127). The supplied caption establishes tissue staining with A03852-3 but does not map its epitope or establish isoform or phosphorylation specificity (caption A03852-3). Therefore, report staining as REST immunoreactivity unless epitope mapping and suitable variant controls support a narrower claim (standard IHC practice). When an isoform-specific interpretation matters, compare antibody staining with an independent assay that resolves the relevant transcript or protein variant in matched material (standard IHC practice).
How should I assess REST in a multiplex IF experiment?
For the separate IF workflow, pair REST with a marker for the expected cell type and a nuclear counterstain so nuclear signal can be assigned to individual cells (UniProt Q13127 localisation; standard IF practice). Select fluorophores after measuring tissue autofluorescence in unstained controls, and place the weaker target signal in a channel with lower background (standard IF practice). REST has no annotated transmembrane segment and is mainly nucleoplasmic, so choose permeabilisation that allows antibody access to the nucleus while preserving morphology (UniProt Q13127 topology; HPA subcellular; standard IF practice). Validate antibody specificity and spectral separation with single-stain and secondary-only controls before interpreting colocalisation (standard IF practice).
How do I reduce diffuse or nonspecific chromogenic staining?
The selected paraffin-section example used 10% goat serum blocking, an overnight 4°C primary incubation at 2 μg/ml, and peroxidase-based DAB detection (caption A03852-3). For high background, compare a no-primary control, check washing and antibody concentration, and keep development time consistent across sections (standard IHC practice). Include an endogenous peroxidase block before HRP detection and inspect any pigment or precipitate that remains in control sections (standard IHC practice). Judge improvement by clearer nuclear signal relative to surrounding tissue, since REST staining is generally nuclear in tissue IHC (HPA tissue IHC; standard IHC practice).
What is a defensible way to quantify REST IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then record nuclear percent-positive cells and staining intensity for an H-score (standard IHC practice; HPA tissue IHC). Calculate the H-score as the sum of each intensity category multiplied by its percentage of cells, using the same scoring thresholds across specimens (standard IHC practice). Normalise counts to the number of eligible cells, or report positive-cell density per mm² when tissue area is the relevant denominator (standard IHC practice). Report cytoplasmic staining separately because REST is mainly nucleoplasmic with additional cytosolic localisation (HPA subcellular).
How can I separate genuine REST signal from staining artefacts?
A convincing positive pattern includes nuclear staining in identifiable cells, consistent with REST's general nuclear tissue profile and mainly nucleoplasmic location (HPA tissue IHC; HPA subcellular). Check cell identity against the section morphology: high signal is reported in bronchial respiratory epithelium, whereas glial cells in cerebral cortex are reported as low (HPA tissue IHC). Treat staining confined to section edges, necrotic regions, or structures positive in a no-primary control as suspect, and examine peroxidase-block controls for endogenous enzyme signal (standard IHC practice). Do not equate absent nuclear staining in every neuron with antibody failure, because REST localisation and abundance vary by context (UniProt Q13127 tissue specificity and localisation).
Boster reagents
Best REST / RE1-silencing transcription factor IHC Antibodies
One human-reactive anti-REST antibody has real IHC data from paraffin-embedded colon, colon cancer and thyroid cancer sections (catalog: A03852-3 reactivity; A03852-3 IHC captions).
Real IHC data
Anti-REST Antibody ®
Cat # A03852-3
A03852-3 is listed for human IHC (catalog: A03852-3 applications/reactivity). Its captions document staining in paraffin-embedded human colon, colon cancer and thyroid cancer sections; no IF figure is supplied (A03852-3 IHC/IF image captions).
Which to pick: For tissue IHC, choose A03852-3, a rabbit antibody listed for human IHC at 2–5 μg/ml (catalog: A03852-3 host/applications/reactivity/dilution); its captions document paraffin sections with EDTA pH 8 retrieval (A03852-3 IHC captions). The fixative is unreported (A03852-3 IHC captions). For IF/ICC or cross-species work, the payload provides no validated option: A03852-3 has no listed IF/ICC application and is human-reactive only (catalog: A03852-3 applications/reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13127 (REST_HUMAN, RE1-silencing transcription factor).
- Human Protein Atlas. REST tissue IHC expression (reliability: Supported).
- Human Protein Atlas. REST subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. REST antibody validation summary (2 antibodies).
- Neuroendocrine Key Regulator Gene Expression in Merkel Cell Carcinoma. Neoplasia (New York, N.Y.) 2018 — PMC6226622.
- Mild Inactivation of RE-1 Silencing Transcription Factor (REST) Reduces Susceptibility to Kainic Acid-Induced Seizures. Frontiers in cellular neuroscience 2019 — PMC6965066.
- REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature. Oncotarget 2018 — PMC5797046.
- Comprehensive Analysis of REST/NRSF Gene in Glioma and Its ceRNA Network Identification. Frontiers in medicine 2021 — PMC8631926.
- PubMed PMID:7697725 — UniProt-cited evidence.
- PubMed PMID:7871435 — UniProt-cited evidence.
- PubMed PMID:8568247 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for REST (RE1-silencing transcription factor). Expected localisation Nuclear tissue staining (HPA tissue IHC); cytoplasm also annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03852-3). Validated IHC antibody: A03852-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial 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 colon cancer tissue; fixative not specified (datasheet A03852-3); 2. Fixation: Image fixative and duration unreported (datasheet A03852-3); 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 A03852-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03852-3); 8. Primary antibody: Rabbit anti-REST, 2-5 μg/ml (datasheet A03852-3); 9. Primary incubation: Overnight at 4 °C (datasheet A03852-3). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03852-3); Caveat: Neuronal nuclear REST can decrease in dementia (UniProt); Isoform / epitope: Four isoforms; validate antibody coverage of each (UniProt); Regulation: Hypoxia increases nuclear REST (UniProt). Evidence: UniProt Q13127, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A03852-3, PMC6226622, PMC5797046. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/REST/immunohistochemistry-ihc-guide.