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TRMT61B / tRNA (adenine(58)-N(1))-methyltransferase, mitochondrial · IHC design guide
Design Immunohistochemistry for TRMT61B
Plan chromogenic IHC on paraffin sections with the catalog antibody A12682-2 (datasheet A12682-2). Compare the observed cytoplasmic staining (HPA tissue IHC) with the protein’s mitochondrial matrix localization (UniProt), using HPA-reported high and low staining tissues to guide controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TRMT61B 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); mitochondrial matrix localization (UniProt) | |
| Staining pattern | Cytoplasmic across diverse cells; high in kidney tubules (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A12682-2) | |
| Positive control | Adrenal gland+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 A12682-2); verify before use. | |
| Caveat | Low staining in adipocytes despite the ubiquitous profile (HPA tissue IHC) | |
| Regulation | Target-specific regulation not annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; epitope map not provided (UniProt) |
Section 1
Recommended TRMT61B IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published pancreatic cancer IHC protocol (PMC13311301).
| Sample | Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet A12682-2) |
| Fixation | Image fixative and duration unreported (datasheet A12682-2); 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 A12682-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A12682-2) |
| Primary antibody | Rabbit anti-TRMT61B, 2-5 μg/ml (datasheet A12682-2) |
| Primary incubation | Overnight at 4 °C (datasheet A12682-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A12682-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TRMT61B-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A12682-2); the published protocol reports retrieval without specifying conditions (PMC13311301: methods).
Section 2
What Is the Expected TRMT61B Staining Pattern?
TRMT61B should appear as cytoplasmic staining in paraffin sections, consistent with its mitochondrial matrix location and lack of a transmembrane segment (HPA: ubiquitous cytoplasmic expression; UniProt Q9BVS5: mitochondrial matrix, no transmembrane segment). Stronger staining is reported in glandular cells, neurons, kidney tubules, and Leydig cells (HPA: High). HPA rates tissue IHC reliability Enhanced, with medium consistency against RNA data and external verification pending (HPA: tissue IHC).
| Cytoplasmic staining is clear in kidney tubular cells or duodenal glandular cells. | This matches reported high staining in both cell types (HPA: High in kidney tubules and duodenal glandular cells). A mitochondrial matrix protein can appear cytoplasmic at the scale of routine chromogenic IHC; the slide alone does not establish precise organelle colocalization (UniProt Q9BVS5: mitochondrial matrix; general IHC interpretation). |
| Staining is confined to nuclei, with little convincing cytoplasmic signal. | A nuclear-only pattern conflicts with the reported cytoplasmic tissue profile and mitochondrial matrix annotation (HPA: ubiquitous cytoplasmic expression; UniProt Q9BVS5: mitochondrial matrix). Assess the counterstain, detection background, and antibody specificity before calling the nuclear signal TRMT61B (general IHC practice). |
| A striking signal appears in a cell population expected to stain weakly, while an adjacent reported high-staining population is faint. | For example, strong adipocyte staining alongside faint kidney tubular staining would conflict with the supplied tissue observations (HPA: Low in adipocytes; High in kidney tubular cells). This raises concern for cross-reactivity or detection background; it does not by itself identify which component caused the signal (general IHC interpretation). |
| Brown color spreads across tissue, edges, or cell-free areas without a readable cytoplasmic pattern. | Diffuse deposit is difficult to assign to TRMT61B because the reported tissue pattern is cellular and cytoplasmic (HPA: ubiquitous cytoplasmic expression). Compare a matched negative detection control and inspect section edges; nonspecific deposition or endogenous detection activity can produce misleading color (general chromogenic IHC practice). |
| A known high-staining tissue shows no cellular signal. | Absence of staining in kidney tubular or adrenal glandular cells conflicts with the reported high signal there (HPA: High in kidney tubular and adrenal glandular cells). Check section integrity and the staining run before interpreting another tissue as negative; HPA's Enhanced rating still has external verification pending (HPA: tissue IHC; general IHC practice). |
💡Expected TRMT61B appearanceCall a result positive when distinct cytoplasmic staining is visible in a reported high-staining cell population, such as kidney tubular cells (HPA: High in kidney tubules; ubiquitous cytoplasmic expression); isolated nuclear color or diffuse cell-free deposit is suspect (UniProt Q9BVS5: mitochondrial matrix; general IHC interpretation).
| Compartment and topology (UniProt Q9BVS5) | TRMT61B is annotated in the mitochondrial matrix and has no transmembrane segment (UniProt Q9BVS5). Interpret a chromogenic cytoplasmic pattern in light of that location; apparent nuclear-only staining needs independent scrutiny (HPA: ubiquitous cytoplasmic expression; general IHC interpretation). |
| Tissue comparison (HPA: tissue IHC) | Kidney tubular, duodenal glandular, neuronal, and Leydig cells are reported High, while adipocytes, vaginal squamous cells, and splenic red-pulp cells are Low (HPA: tissue IHC). Use those differences as comparison points; Low does not mean antigen-free (HPA: Low). |
| Antibody evidence (HPA: antibody validation) | HPA026747 and HPA026751 each have Enhanced IHC status (HPA: antibody validation). The tissue profile has medium consistency with RNA expression and awaits external verification (HPA: tissue IHC), so a plausible staining pattern remains evidence to evaluate rather than proof of specificity. |
| IF/ICC Q: should the signal be mitochondrial? | UniProt places TRMT61B in the mitochondrial matrix, while HPA reports cytosol as an uncertain ICC-IF location (UniProt Q9BVS5: subcellular location; HPA: subcellular ICC-IF). Treat that difference as unresolved; this IHC pattern cannot settle the IF/ICC localization question. |
| Fixation sensitivity and retrieval | Target-specific fixation sensitivity and a TRMT61B retrieval condition are not supplied (UniProt Q9BVS5; HPA: tissue IHC). If a run fails, review the documented antibody method and controls before adjusting retrieval; any such adjustment is general IHC optimization, not an established TRMT61B requirement. |
| Situation | Likely cause | Next action |
|---|---|---|
| High-staining reference tissue is blank. | The result conflicts with reported high staining in kidney tubular or duodenal glandular cells (HPA: High). An unsuccessful staining run remains possible (general IHC practice). | Confirm the section contains the expected cells, inspect run controls, and check the documented antibody dilution, retrieval, and detection steps before scoring the test section negative (general IHC practice). |
| Signal is nuclear-only. | The compartment disagrees with the cytoplasmic IHC profile and mitochondrial matrix annotation (HPA: ubiquitous cytoplasmic expression; UniProt Q9BVS5: mitochondrial matrix). | Review counterstain and a matched negative detection control, then compare with cytoplasmic staining in a reported high-staining tissue (general IHC practice; HPA: High in kidney tubules). |
| The entire section has diffuse brown haze. | A tissue-wide deposit obscures the reported cellular cytoplasmic pattern (HPA: ubiquitous cytoplasmic expression). Background from detection chemistry is a possible explanation (general chromogenic IHC practice). | Inspect a matched negative detection control and review blocking, washes, and chromogen development against the documented method (general chromogenic IHC practice). |
| Low-reported cells stain more strongly than nearby high-reported cells. | That ordering conflicts with the supplied comparison, such as Low adipocytes versus High kidney tubular cells (HPA: tissue IHC). Cross-reactivity or endogenous detection activity may contribute (general IHC interpretation). | Verify cell identity and compare the same cell populations across controls and another section; investigate the detection control before assigning the unexpected color to TRMT61B (general IHC practice). |
| Signal is present but cell boundaries or cytoplasm cannot be judged. | Dense chromogen or a strong counterstain can make compartment scoring unreliable (general chromogenic IHC practice). The expected tissue profile is cytoplasmic (HPA: ubiquitous cytoplasmic expression). | Review development and counterstain against the documented method, then reassess whether color lies within the reported high-staining cell population (general IHC practice; HPA: High in kidney tubules). |
| Two antibodies give different tissue patterns. | Enhanced IHC status applies to both HPA026747 and HPA026751, but the reported tissue profile still awaits external verification (HPA: antibody validation; HPA: tissue IHC). | Compare matched sections, controls, and the documented conditions for each antibody; record the disagreement instead of treating either pattern alone as definitive (general IHC practice; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing TRMT61B staining
A12682-2 · IHCIHC analysis of TRMT61B using anti-TRMT61B antibody (A12682-2). TRMT61B was detected in a paraffin-embedded section of human colorectal aden
A12682-2 · IHCIHC analysis of TRMT61B using anti-TRMT61B antibody (A12682-2). TRMT61B was detected in a paraffin-embedded section of human liver cancer ti
A12682-2 · IFIF analysis of TRMT61B using anti-TRMT61B antibody (A12682-2). TRMT61B was detected in a paraffin-embedded section of human intestinal cance
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000171103-TRMT61B/tissue/cerebral+cortex#imid_6660568
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000171103-TRMT61B/tissue/colon#imid_6660486
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000171103-TRMT61B/tissue/liver#imid_6660487
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000171103-TRMT61B/tissue/kidney#imid_6660480
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026751; green = TRMT61B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA026751; green = TRMT61B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TRMT61B IHC & IF
🧪Run cerebral cortex first and score neuronal cells, which show high TRMT61B staining (HPA: High in cerebral cortex neuronal cells). HPA detects TRMT61B in all 45 scored tissues, so there is no documented negative tissue; use no-primary and isotype controls for background, and compare non-neuronal cells on the positive slide without assuming they are TRMT61B-negative (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: TRMT61B 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 TRMT61B in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype-matched control appropriate to the primary antibody’s host species; confirm signal specificity with TRMT61B knockout material where available (standard IHC practice). For chromogenic IHC, quench endogenous peroxidase and check the cerebral cortex for pigment that could resemble staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A12682-2 paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption). That caption uses heat retrieval in EDTA at pH 8.0, but it does not establish whether retrieval is required for every specimen or whether frozen sections are easier (selected-SKU tissue-IHC caption). IF/ICC feasibility needs separate assessment because HPA reports uncertain cytosolic localization in its cell images while UniProt places TRMT61B in the mitochondrial matrix; cerebral cortex neuronal lipofuscin can also create autofluorescence (HPA subcellular; UniProt Q9BVS5 localization; standard IF practice).
HPA tissue IHC evidence for TRMT61B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: TRMT61B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced TRMT61B IHC Tips
Use the selected paraffin section workflow as the IHC starting point, then assess staining against TRMT61B localisation and controls.
What retrieval should I try first if TRMT61B staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A12682-2). Keep heating and cooling consistent across slides, then vary retrieval duration on matched sections while checking tissue architecture (standard IHC practice). The selected image used 2 μg/ml primary antibody overnight at 4°C; hold those conditions constant during the first retrieval comparison (datasheet A12682-2). If staining remains weak, test citrate buffer at pH 6.0 as a fallback and record each heating time (standard IHC practice). Compare cellular signal, morphology and a no-primary control before selecting a condition (standard IHC practice).
How should I troubleshoot variable TRMT61B staining between paraffin blocks?
TRMT61B-specific fixation sensitivity is unknown because the selected paraffin section caption does not state its fixative (datasheet A12682-2). Record the fixative, fixation duration and processing history for each block before comparing staining intensity (standard IHC practice). For prospectively collected material, 10% neutral buffered formalin is a routine starting fixative; keep its duration consistent and document it (standard IHC practice). Stain matched sections in the same run using EDTA at pH 8.0 and the same detection conditions (datasheet A12682-2; standard IHC practice). If blocks still differ, assess morphology and technical controls before attributing intensity differences to TRMT61B abundance (standard IHC practice).
Should TRMT61B appear mitochondrial or diffusely cytoplasmic in chromogenic IHC?
TRMT61B is annotated in the mitochondrial matrix and has no transmembrane segment (UniProt Q9BVS5 localisation and topology). At light-microscope resolution, examine whether cytoplasmic DAB staining has a granular distribution, but avoid claiming matrix localisation from chromogenic staining alone (standard IHC practice). HPA reports ubiquitous cytoplasmic tissue staining while its ICC/IF cytosol assignment is uncertain, so that evidence does not settle the compartment discrepancy (HPA tissue IHC; HPA subcellular). Compare staining within a named cell population, such as kidney tubular cells, which HPA rates high (HPA: High in kidney tubular cells). Unexpected nuclear-only staining warrants specificity controls and review of the counterstain (UniProt Q9BVS5 localisation; standard IHC practice).
Could an isoform or hidden epitope explain absent TRMT61B staining?
No alternative isoforms are annotated for the 477-amino-acid TRMT61B precursor, so an isoform explanation lacks support in this record (UniProt Q9BVS5 processing and isoforms). The record also annotates no glycosylation sites, modified residues or transmembrane segment (UniProt Q9BVS5 annotations). The antibody’s exact epitope is unspecified, so do not assume whether retrieval exposes it or whether processing changes antibody binding (datasheet A12682-2; UniProt Q9BVS5 processing). First repeat the documented EDTA retrieval at pH 8.0 with a positive control section (datasheet A12682-2; standard IHC practice). If available, compare a second antibody with a documented, nonoverlapping epitope on adjacent sections (standard IHC practice).
How can IF help assess the localisation seen in TRMT61B IHC?
Use IF as a separate localisation check: UniProt places TRMT61B in the mitochondrial matrix, whereas HPA’s ICC/IF cytosol assignment is uncertain (UniProt Q9BVS5 localisation; HPA subcellular). Multiplex with a mitochondrial marker and a marker identifying the expected cell population, such as kidney tubular cells, then inspect both cellular identity and signal overlap (HPA: High in kidney tubular cells; standard IF practice). Choose spectrally separated fluorophores and assess tissue autofluorescence before interpreting weak signal, particularly in shorter wavelength channels (standard IF practice). Because a matrix epitope lies behind the plasma and mitochondrial membranes, titrate permeabilisation while checking mitochondrial morphology; the antibody epitope is unspecified (UniProt Q9BVS5 localisation; datasheet A12682-2; standard IF practice).
What should I check when TRMT61B DAB staining is widespread?
The selected paraffin section workflow blocked with 10% goat serum, used a peroxidase-conjugated secondary and developed with DAB (datasheet A12682-2). Include a no-primary section to reveal secondary or detection background, and apply an endogenous peroxidase block as a general chromogenic IHC step (standard IHC practice). Keep primary concentration at the documented 2 μg/ml while first checking wash consistency and the 30-minute secondary incubation (datasheet A12682-2; standard IHC practice). Examine whether staining follows tissue edges, folds or damaged regions rather than intact cells (standard IHC practice). Broad cytoplasmic staining needs cautious interpretation because HPA describes ubiquitous cytoplasmic expression (HPA tissue IHC).
How should I score TRMT61B staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable analysis area before scoring, and use identical staining and imaging conditions across sections (standard IHC practice). For cellular intensity, an H-score combines the percentages of cells at intensities 0–3 into a score of 0–300 (standard IHC practice). Alternatively, report the percentage of positive eligible cells, or positive-cell density per mm² of viable tissue (standard IHC practice). Normalise cell-based scores to all eligible cells in the same compartment and area-based density to measured viable area (standard IHC practice). Stratify by cell type because HPA rates kidney tubular cells high and adipocytes low (HPA tissue IHC).
How can I distinguish plausible TRMT61B staining from artefact?
Interpret cellular staining alongside the mitochondrial matrix annotation and HPA’s ubiquitous cytoplasmic tissue profile, recognising that DAB cannot establish matrix residence by itself (UniProt Q9BVS5 localisation; HPA tissue IHC; standard IHC practice). HPA rates kidney tubular cells high and adipocytes low, providing contrasting populations for a staining review (HPA tissue IHC). Strong signal confined to nuclei, tissue edges, folds or necrotic areas should prompt scrutiny before scoring (UniProt Q9BVS5 localisation; standard IHC practice). Check a no-primary section and the endogenous peroxidase block when DAB appears in unexpected structures (standard IHC practice). Treat agreement with expected location and cell populations as supportive evidence, then seek an independent specificity control (standard IHC practice).
Boster reagents
Best TRMT61B / tRNA (adenine(58)-N(1))-methyltransferase, mitochondrial IHC Antibodies
A12682-2 has human paraffin-section IHC images and an IF image from a human paraffin section (catalog image captions).
Real IHC data
Anti-TRMT61B Antibody ®
Cat # A12682-2
A12682-2 is listed for human IHC and IF (catalog: applications/reactivity). Its IHC images show human paraffin sections of colorectal adenocarcinoma, liver cancer, ovarian cancer, and spleen; its IF image shows a human intestinal cancer paraffin section (A12682-2 image captions).
Which to pick: For tissue IHC, choose A12682-2 for human paraffin sections: its own IHC captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A12682-2 IHC image captions). For IF, A12682-2 has a human intestinal cancer paraffin-section image using 5 μg/ml; ICC validation is unreported (A12682-2 IF image caption; catalog applications). No cross-species or clonality-based choice is supported: A12682-2 lists only human reactivity and has no clone designation (catalog: reactivity/clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BVS5 (TR61B_HUMAN, tRNA (adenine(58)-N(1))-methyltransferase, mitochondrial).
- Human Protein Atlas. TRMT61B tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TRMT61B subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. TRMT61B antibody validation summary (2 antibodies).
- Identification and validation of an m1A-score model to predict outcomes and immunomodulation in lung squamous cell carcinoma by integrated analysis of single-cell and bulk RNA sequencing. Discover oncology 2026 — PMC12936309.
- Molecular Subtype Identification and Prognostic Prediction of Pancreatic Cancer Based on m6A/m5C/m1A-Related Genes. Journal of cellular and molecular medicine 2026 — PMC13311301.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TRMT61B (tRNA (adenine(58)-N(1))-methyltransferase, mitochondrial). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial matrix localization (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A12682-2). Validated IHC antibody: A12682-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular 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 colorectal adenocarcinoma tissue; fixative not specified (datasheet A12682-2); 2. Fixation: Image fixative and duration unreported (datasheet A12682-2); 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 A12682-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A12682-2); 8. Primary antibody: Rabbit anti-TRMT61B, 2-5 μg/ml (datasheet A12682-2); 9. Primary incubation: Overnight at 4 °C (datasheet A12682-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A12682-2); verify before use.; Caveat: Low staining in adipocytes despite the ubiquitous profile (HPA tissue IHC); Isoform / epitope: No annotated isoforms; epitope map not provided (UniProt); Regulation: Target-specific regulation not annotated (UniProt). Evidence: UniProt Q9BVS5, HPA tissue IHC · Enhanced, HPA ICC-IF · cytosol., Datasheet A12682-2, PMC13311301. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TRMT61B/immunohistochemistry-ihc-guide.