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RTL6 / Retrotransposon Gag-like protein 6 · IHC design guide
Design Immunohistochemistry for RTL6
Plan RTL6 chromogenic IHC on paraffin sections with the catalog antibody at 1:100–1:300 (datasheet). Compare kidney tubule staining, reported as high, with adipocytes, reported as not detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RTL6 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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal across most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | No expression regulation annotated (UniProt) | |
| Isoform / epitope | No isoforms; one 1–239 chain (UniProt) |
Section 1
Recommended RTL6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by one published tissue microarray IHC protocol (PMC13048935).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A31816) |
| Fixation | Image fixative and duration unreported (datasheet A31816); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-RTL6, 1:100-1:300 (datasheet A31816) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RTL6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval rule); the published excerpt does not specify retrieval (PMC13048935: methods).
Section 2
What Is the Expected RTL6 Staining Pattern?
For paraffin-section IHC, expect predominantly cytoplasmic RTL6 staining in many tissues, with high staining reported in kidney tubule cells, cerebellar Purkinje cells and several other defined cell populations (HPA tissue IHC). RTL6 has no annotated transmembrane segment or subcellular location (UniProt Q6ICC9). Treat the tissue pattern as a guide rather than proof of specificity: HPA rates its IHC evidence Approved but reports low consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct cytoplasmic chromogen in kidney tubule cells or cerebellar Purkinje cells, with surrounding structures still distinguishable. | This matches the reported compartment and two high-staining cell populations (HPA tissue IHC). Score the fraction and intensity of stained cells within each population; a convincing positive result retains cellular boundaries. HPA's Approved rating supports using this pattern as a reference, while its low antibody–RNA consistency limits how confidently staining alone identifies RTL6 (HPA tissue IHC). |
| Predominantly nuclear or membrane-rim staining in an IHC section, with little discernible cytoplasmic signal. | Recheck interpretation and controls before calling this RTL6: the tissue IHC profile is cytoplasmic (HPA tissue IHC), and UniProt annotates no transmembrane segment (UniProt Q6ICC9 topology). Nuclear speckles are approved in HPA ICC-IF, so a nuclear signal alone does not prove an artefact; the assay-specific discrepancy needs investigation (HPA ICC-IF). |
| Strong staining in adipocytes or soft-tissue fibroblasts, especially when expected positive cells are faint. | Those populations were reported as not detected (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic detection activity as possible explanations, then examine negative and detection controls. A negative HPA observation is a comparator, not proof that every specimen of that tissue must be blank (HPA tissue IHC). |
| Similar diffuse colour across cells, extracellular areas and tissue edges, without a defined cytoplasmic pattern. | Treat this as background until controls establish otherwise. Check whether the same colour appears without primary antibody; if so, investigate the detection system, blocking and washing as general IHC troubleshooting steps. The diffuse distribution does not resemble HPA's predominantly cytoplasmic tissue profile (HPA tissue IHC). |
| No signal in an adequately preserved kidney-tubule or Purkinje-cell positive-control section. | Both cell populations are reported as High (HPA tissue IHC). First check that the expected cells are present, then review the catalog antibody's IHC-P instructions and detection controls. Absence of signal raises a technical or specimen question; it cannot establish biological absence from one failed section. |
💡Expected RTL6 appearanceCall a positive IHC result when defined cells show clear, predominantly cytoplasmic chromogen, potentially strong in kidney tubules or Purkinje cells (HPA tissue IHC); uniform haze or strong staining in HPA-not-detected adipocytes is suspect (HPA tissue IHC).
| Tissue and cell selection | HPA reports High staining in adrenal, seminal-vesicle and stomach glandular cells; kidney tubules; Purkinje and cortical neuronal cells; lymph-node germinal-center cells; and late spermatids (HPA tissue IHC). Its low-tissue-specificity RNA classification means tissue identity alone is a weak specificity test (HPA tissue IHC). |
| Antibody evidence | HPA005697 is rated Approved for IHC, while the tissue profile notes low consistency between antibody staining and RNA data (HPA antibodies; HPA tissue IHC). Interpret morphology together with controls and avoid treating the staining score as an independently verified measure of RTL6 abundance. |
| Topology and processing | UniProt lists a single 1–239 chain, no signal peptide, no propeptide, no transmembrane segment and no annotated glycosylation sites (UniProt Q6ICC9). These annotations do not identify the antibody epitope or predict antigen-retrieval conditions. |
| Antigen retrieval | Use the catalog antibody's IHC-P instructions to choose initial retrieval conditions; the supplied HPA and UniProt records give no RTL6-specific retrieval condition or fixation-sensitivity result. If optimisation is needed, compare conditions with the same positive tissue and controls as general IHC practice. |
| IF/ICC Q: Should its nuclear speckles change the IHC call? | A: HPA ICC-IF places RTL6 mainly in nuclear speckles and additionally in cytosol, whereas HPA tissue IHC describes cytoplasmic staining (HPA ICC-IF; HPA tissue IHC). Interpret each assay against its own evidence; a compartment difference merits validation rather than an automatic positive or negative call. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-control kidney section is blank. | The sampled section may lack identifiable tubules, or the IHC staining workflow may have failed; kidney tubule cells are High in the reference profile (HPA tissue IHC). | Confirm tubules on the counterstain, inspect the detection control and follow the catalog antibody's IHC-P instructions. Repeat with a suitable positive section before interpreting study samples; do not assign an RTL6-negative score from the failed run. |
| Every tissue shows similar diffuse colour. | Background from detection reagents, inadequate blocking or insufficient washing is plausible in chromogenic IHC; this appearance differs from the defined cytoplasmic HPA profile (HPA tissue IHC). | Compare a no-primary control and examine whether colour follows cells or tissue edges. Review blocking, wash steps and detection chemistry as general IHC practice, then rescore only when cell-specific signal can be separated from background. |
| Adipocytes or fibroblasts stain strongly. | These cells are reported as Not detected (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible, although HPA's observation cannot exclude genuine differences in another specimen. | Check staining in a no-primary control, examine morphology and compare with an HPA High population processed in the same run (HPA tissue IHC). If the discordance persists, report it as unresolved rather than assigning RTL6 positivity. |
| The only convincing IHC signal is nuclear. | The result conflicts with HPA's cytoplasmic tissue IHC profile, although HPA ICC-IF independently reports approved nuclear-speckle localisation (HPA tissue IHC; HPA ICC-IF). | Verify that nuclei and cytoplasm are correctly identified with the counterstain, inspect background controls and document the compartment separately. Do not transfer the ICC-IF localisation directly into an IHC scoring rule. |
| Low signal appears in prostate or colon glandular cells. | Low staining is reported for those glandular populations (HPA tissue IHC); a weak result there does not by itself show assay failure. | Judge run performance in a clearly represented High population, such as kidney tubules or stomach glandular cells (HPA tissue IHC). Score the low population separately, using the same background threshold across sections. |
| Changing retrieval gives a different staining score. | The supplied records do not establish an RTL6-specific retrieval response or fixation effect; technical conditions can change chromogenic IHC appearance as a general practice consideration. | Compare matched sections, positive tissue and no-primary controls under the tested conditions. Record the chosen condition and its control performance without claiming that HPA tissue levels or UniProt topology explain the retrieval difference. |
Experiment evidence
IHC & IF images showing RTL6 staining
A31816 · IHCImmunohistochemistry validation of RTL6 using Anti-LDOC1L RTL6 Antibody (A31816). Immunohistochemical analysis of paraffin-embedded Human br
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000188636-RTL6/tissue/cerebral+cortex#imid_2290200
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000188636-RTL6/tissue/colon#imid_2290134
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000188636-RTL6/tissue/liver#imid_2290127
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000188636-RTL6/tissue/kidney#imid_2290128
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA005697; green = RTL6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA005697; green = RTL6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RTL6 IHC & IF
🧪Run cerebellum first and look for staining in Purkinje cells (HPA: High in Purkinje cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); cells without specific signal on the cerebellum slide should show only background chromogen, but HPA does not identify a specific cerebellar cell type as negative (HPA: cerebellum row).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RTL6 in A-431, U-251MG, U2OS, with annotated localisation: Nuclear speckles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and, where available, RTL6 knockout tissue or a validated immunogen-peptide block (standard IHC practice). Block endogenous peroxidase and distinguish brain pigment from chromogen when assessing the cerebellum slide (standard chromogenic brain IHC practice).
⚠️Feasibility: A target-specific fixation window is unreported, and the selected A31816 paraffin-section caption does not state a fixative (selected-SKU tissue-IHC caption). Retrieval dependence is unreported in the supplied evidence; assess retrieval with the catalog antibody’s IHC-P protocol (selected-SKU tissue-IHC caption; standard IHC practice). The evidence does not establish that frozen sections or IF are easier; brain pigment can complicate chromogenic scoring, while IF requires attention to tissue autofluorescence (standard IHC/IF practice).
HPA tissue IHC evidence for RTL6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
Section 3
Advanced RTL6 IHC Tips
Use RTL6 compartment and cell-type patterns to assess paraffin-section staining, while treating antibody performance and fixation sensitivity as separate validation questions.
What retrieval should I start with for RTL6 in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule: cytoplasmic or membrane antigen). Let slides cool in the retrieval buffer, then compare staining with a matched section processed without retrieval to reveal heat-dependent background (standard IHC practice). The selected antibody has a paraffin-embedded human brain IHC image, but its caption does not report retrieval conditions or a fixative (A31816 caption). If staining remains weak, test a more alkaline retrieval buffer on adjacent sections as a fallback, holding detection and exposure conditions constant (standard IHC optimization).
How should I investigate weak RTL6 staining when fixation history varies?
Target-specific fixation sensitivity is unknown: the selected paraffin-embedded human brain caption does not state its fixative (A31816 caption). Record fixation duration and processing history for each block, and compare adjacent sections using the same citrate pH 6.0, 95–98 °C, 20 min retrieval and detection run (page retrieval rule; standard IHC practice). Use a previously staining control section to distinguish a batch problem from a specimen-specific loss of signal (standard IHC practice). Do not attribute differences to RTL6 fixation sensitivity from its tissue staining pattern, lack of annotated modifications, or lack of a transmembrane segment (HPA tissue IHC; UniProt Q6ICC9).
How should I assess cytoplasmic versus nuclear RTL6 staining?
Assess the fraction of stained cells and record cytoplasmic and nuclear signals separately at a consistent magnification (standard IHC practice). HPA describes cytoplasmic expression in most tissues by tissue IHC, while its ICC/IF data place RTL6 mainly in nuclear speckles with additional cytosolic localization (HPA tissue IHC; HPA subcellular). These observations support examining both compartments, but a fine speckled nuclear pattern may be difficult to resolve with a chromogenic counterstain (HPA subcellular; standard IHC practice). Compare adjacent 3–5 µm sections stained and counterstained together, and report the observed compartment rather than assigning one as universally correct (standard IHC practice).
Can an unexpected RTL6 pattern reflect an isoform or inaccessible epitope?
UniProt lists one 1–239 RTL6 chain and no annotated isoforms, modified residues, or glycosylation sites (UniProt Q6ICC9). Those annotations do not identify the catalog antibody’s epitope or establish its accessibility after paraffin processing (UniProt Q6ICC9; A31816 caption). If staining changes after citrate pH 6.0 retrieval, compare adjacent sections with identical antibody dilution and detection to isolate the retrieval effect (page retrieval rule; standard IHC practice). Check the antibody’s documented immunogen or epitope information before interpreting compartment differences as distinct RTL6 forms, and seek independent antibody validation where possible (standard IHC validation practice).
How can IF help evaluate an ambiguous RTL6 IHC pattern?
Use IF as a separate validation experiment, then compare its cell-level pattern with adjacent chromogenic IHC sections (standard IHC/IF practice). Multiplex RTL6 with a marker for the expected cell type, such as a neuronal marker when examining the brain image context, and include single-label controls (A31816 caption: human brain; standard IF practice). Choose a far-red RTL6 fluorophore when tissue autofluorescence interferes with shorter wavelengths, and inspect unstained sections first (standard IF practice). For an intracellular epitope, test gentle permeabilization such as 0.1% detergent for 5 min; the precise epitope location is unreported, although RTL6 has no annotated transmembrane segment (UniProt Q6ICC9; standard IF practice).
What controls separate RTL6 signal from chromogenic background?
Run a no-primary control and inspect unstained tissue before interpreting diffuse brown deposits as RTL6 (standard IHC practice). Block endogenous peroxidase before HRP/DAB detection, and compare sections developed for the same 2–5 min interval so development time cannot explain apparent differences (standard chromogenic IHC practice). HPA reports cytoplasmic staining in most tissues, which makes diffuse signal alone a weak specificity check (HPA tissue IHC). Contrast the expected high staining in cerebellar Purkinje cells with reported nondetection in adipocytes or soft-tissue fibroblasts, while remembering that HPA rates agreement between staining and RNA expression as low (HPA tissue IHC).
How should I quantify RTL6 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell types and regions before scoring, and keep section thickness, retrieval, detection, and image settings consistent (standard IHC quantification practice). For a chromogenic cell-based result, report percentage of positive cells and an H-score using intensity categories 0–3, calculated as the sum of each category’s percentage multiplied by its intensity (standard IHC scoring practice). Normalize positive-cell counts to all evaluable cells of the same type, or report positive cells per mm² of viable tissue when cell boundaries are uncertain (standard IHC quantification practice). Score cytoplasmic and nuclear staining separately, since tissue IHC and ICC/IF report different dominant RTL6 compartments (HPA tissue IHC; HPA subcellular).
Which RTL6 staining patterns warrant skepticism before calling a true positive?
Treat reproducible staining in the expected cell population and compartment as stronger evidence than isolated dark deposits (standard IHC interpretation practice). HPA reports high staining in cerebellar Purkinje cells and cerebral-cortex neurons, with broad cytoplasmic tissue staining; its ICC/IF data also support nuclear speckles and cytosol (HPA tissue IHC; HPA subcellular). Inspect section edges, necrotic regions, and no-primary controls for edge effects, trapped DAB, or endogenous enzyme signal before scoring (standard IHC practice). Because HPA reports low consistency between antibody staining and RNA expression, confirm a surprising cell-type pattern with an independent antibody or orthogonal assay before assigning it to RTL6 (HPA tissue IHC; standard IHC validation practice).
Boster reagents
Best RTL6 / Retrotransposon Gag-like protein 6 IHC Antibodies
A31816 has a human brain paraffin-section IHC image (catalog IHC caption), lists IF as an application, and lists Human and Mouse reactivity (catalog applications and reactivity).
Real IHC data
Anti-LDOC1L RTL6 Antibody
Cat # A31816
A31816 will render with an IHC image of paraffin-embedded human brain at 1:100 (catalog IHC caption). IF is listed as an application, but no IF image is supplied (catalog applications and image alts).
Which to pick: For tissue IHC, choose A31816: its image shows paraffin-embedded human brain, and the listed IHC dilution is 1:100–1:300 (catalog IHC caption; datasheet: IHC dilution). For IF, A31816 lists a 1:50 dilution; ICC validation and an IF image are unreported (catalog applications, dilution and image alts). A31816 lists Human and Mouse reactivity, but the supplied IHC image shows Human only; the tissue fixative and antibody clonality are unreported (catalog reactivity, IHC caption and antibody fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6ICC9 (RTL6_HUMAN, Retrotransposon Gag-like protein 6).
- Human Protein Atlas. RTL6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RTL6 subcellular location (ICC-IF): Mainly localized to the nuclear speckles. In addition localized to the cytosol..
- Human Protein Atlas. RTL6 antibody validation summary (1 antibodies).
- Multi-modal molecular and spatial profiling reveals NNT as a prognostic biomarker in obesity-associated colorectal cancer. Journal of gastroenterology 2026 — PMC13048935.
- PubMed PMID:15461802 — UniProt-cited evidence.
- PubMed PMID:10591208 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RTL6 (Retrotransposon Gag-like protein 6). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A31816 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human brain tissue; fixative not specified (datasheet A31816); 2. Fixation: Image fixative and duration unreported (datasheet A31816); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-RTL6, 1:100-1:300 (datasheet A31816); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms; one 1–239 chain (UniProt); Regulation: No expression regulation annotated (UniProt). Evidence: UniProt Q6ICC9, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear speckles. In addition localized to…, Datasheet A31816, PMC13048935. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RTL6/immunohistochemistry-ihc-guide.