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MTHFR / Methylenetetrahydrofolate reductase (NADPH) · IHC design guide
Design Immunohistochemistry for MTHFR
Plan MTHFR staining in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC). Compare glandular and kidney tubule cells with documented low or undetected cell populations when assessing signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MTHFR 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) | |
| Staining pattern | Cytoplasmic staining in glandular and kidney tubule cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A00336) | |
| Positive control | Bronchus+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 | Antibody staining has only medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Staining-linked regulation is unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended MTHFR IHC & IF Protocols
The catalog antibody protocol uses heat-mediated Tris-EDTA pH 9.0 retrieval (datasheet A00336). The published IHC options below report tissue staining methods from three MTHFR studies (PMC10290629; PMC7353690; PMC9353118).
| Sample | Paraffin-embedded human oophoroma tissue; fixative not specified (datasheet A00336) |
| Fixation | Image fixative and duration unreported (datasheet A00336); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A00336); 20 min, 95–100 °C (standard) |
| 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-MTHFR, 1:50 recommended; image 1:200 (datasheet A00336) |
| 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 | MTHFR-positive staining in ciliated cells (cilia axoneme) of bronchus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, including epididymis, testis, kidney, anterior pituitary gland, lymphoid and neuronal tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval for the catalog antibody (datasheet A00336); the cited article excerpts do not specify retrieval conditions (PMC10290629; PMC7353690; PMC9353118).
Section 2
What Is the Expected MTHFR Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic MTHFR staining in selected glandular and tubular cells, including epididymal glandular cells and kidney tubule cells (HPA: tissue IHC). Bronchial ciliated cells can show ciliary axoneme staining (HPA: High). Tissue IHC reliability is Enhanced, with medium consistency between staining and RNA data (HPA). UniProt assigns no subcellular location and reports no transmembrane segment (UniProt P42898 topology).
| Clear cytoplasmic staining in kidney tubule cells or epididymal glandular cells, with recognizable tissue structure. | This matches the reported high staining in those cell populations (HPA: kidney and epididymis, High). Score the named cells and their compartment; adjacent unstained cells need not invalidate the result (standard IHC practice). |
| Predominantly nuclear staining, or another unexpected compartment, replaces the expected cytoplasmic pattern. | Treat this as a possible localization artefact and check controls before assigning it to MTHFR (HPA: cytoplasmic tissue IHC profile). UniProt supplies no subcellular annotation that would independently confirm a new compartment (UniProt P42898). |
| Strong staining appears in lung alveolar cells or adipocytes, while expected positive cells stain weakly. | Those cell populations are reported as not detected (HPA: lung alveolar cells; adipose adipocytes). Consider cross-reactivity or endogenous detection activity; compare cell-resolved controls before interpreting the signal (standard IHC practice). |
| Color is spread across stroma, empty spaces or most cells without a discernible cellular pattern. | Diffuse background cannot establish MTHFR localization. Assess reagent-only and tissue background, washing, blocking and chromogen development (standard IHC practice); HPA's cell-specific observations do not validate uniform staining (HPA: tissue IHC). |
| No signal is visible in kidney tubule cells or epididymal glandular cells on an otherwise interpretable section. | This conflicts with reported high staining in those cells (HPA: kidney and epididymis, High). Check whether the assay worked using a known-positive section; absence alone cannot distinguish technical failure from specimen variation (standard IHC practice). |
💡Expected MTHFR appearanceCall a positive result when staining is predominantly cytoplasmic in kidney tubule or epididymal glandular cells at a readily visible level (HPA: High); diffuse, nuclear-only or strongly alveolar staining warrants control checks (HPA: tissue IHC; standard IHC practice).
| Choice of IHC evidence | HPA076180 and HPA077255 carry Enhanced IHC validation (HPA: antibodies); the overall tissue profile has medium staining-to-RNA consistency (HPA: reliability description). Compare the actual slide with the reported cells. |
| Cell population within a tissue | Interpret cells separately: bronchial cilia axonemes are High, whereas lung alveolar cells are not detected (HPA: bronchus and lung). A tissue-level positive label cannot be applied to every cell. |
| Molecular features | UniProt lists two isoforms, no signal peptide or propeptide, and a single chain spanning residues 1–656 (UniProt P42898). These facts do not establish isoform-specific IHC staining or a processing-related compartment change. |
| Detection background | With an enzyme-based chromogen, endogenous tissue activity can mimic antibody staining (standard IHC practice). Use the appropriate enzyme control and assess staining with the primary antibody omitted before assigning atypical color to MTHFR. |
| Q: Should IF/ICC show the same localization as paraffin-section IHC? | A: HPA IF/ICC reports mainly cell junctions, with centrosome and basal-body staining (HPA: subcellular ICC-IF). This is assay-specific evidence; it does not replace the cytoplasmic tissue IHC reference (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | Assay sensitivity or detection may be inadequate; kidney tubule and epididymal glandular cells are reported High (HPA: tissue IHC). | Run a known-positive section alongside the specimen and review antigen retrieval and detection controls (standard IHC practice). No MTHFR-specific retrieval condition or fixation sensitivity is established by these sources. |
| Only the section edge or damaged areas are strongly colored. | Uneven reagent access or local background can distort interpretation (standard IHC practice). | Judge intact interior cells, inspect an antibody-omission control, and repeat with even reagent coverage if needed (standard IHC practice). Do not score edge color as a cell-specific MTHFR pattern. |
| Unexpected nuclear staining dominates. | The pattern disagrees with the reported cytoplasmic tissue profile (HPA: tissue IHC); the source data do not establish a nuclear IHC pattern. | Compare with a positive tissue and an antibody-omission control, then review antibody concentration and detection background (standard IHC practice). Record the discrepancy rather than reclassifying localization. |
| Lung alveolar cells or adipocytes appear strongly positive. | These cells are reported as not detected (HPA: lung alveolar cells; adipose adipocytes); cross-reactivity or endogenous detection activity is possible. | Check cell identity and compare antibody-omission and enzyme-background controls where relevant (standard IHC practice). Interpret discordant staining cautiously, even when another cell population in the section stains. |
| Most cells show weak, uniform chromogen. | Diffuse background may obscure the cell-resolved cytoplasmic pattern reported by HPA (HPA: tissue IHC; standard IHC practice). | Review blocking, washing, primary-antibody concentration and chromogen development with matched controls (standard IHC practice). Reassess named positive and negative cell populations after background is reduced. |
| A negative-control tissue shows little color, but so does the positive tissue. | A clean negative alone does not show that the assay can detect MTHFR (standard IHC practice). HPA reports high staining in kidney tubules and epididymal glandular cells (HPA: tissue IHC). | Confirm that a known-positive section develops cell-specific signal before interpreting absent specimen staining; document the tested cell population and controls (standard IHC practice). |
Experiment evidence
IHC & IF images showing MTHFR staining
A00336 · IHCImmunohistochemical analysis of paraffin-embedded human oophoroma. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was
M00336 · IHCImmunohistochemistry analysis of paraffin-embedded lung cancer tissues with DAB staining using MTHFR Monoclonal Antibody.
HPA · Pituitary glandHuman Protein Atlas IHC, Pituitary gland. Source: https://www.proteinatlas.org/ENSG00000177000-MTHFR/tissue/pituitary+gland#imid_1403
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000177000-MTHFR/tissue/cerebral+cortex#imid_20586683
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000177000-MTHFR/tissue/colon#imid_20588189
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000177000-MTHFR/tissue/liver#imid_20588206
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA063389; green = MTHFR, blue = nucleus, red = microtubules.
HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA063389; green = MTHFR, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MTHFR IHC & IF
🧪Run kidney first: cells in tubules should stain strongly (HPA: Kidney, cells in tubules, High). Use heart muscle as the negative tissue: cardiomyocytes are listed as not detected (HPA: Heart muscle, cardiomyocytes, Not detected); on the kidney slide, assess any cells outside the stained tubules for background rather than assuming a particular cell type is MTHFR-negative.
Positive control tissue: Bronchus (Ciliated cells (cilia axoneme), HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MTHFR in SH-SY5Y, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Cell Junctions (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and clonality-matched isotype controls, plus MTHFR-knockout tissue or a validated peptide-block control (standard IHC practice). In kidney sections, check endogenous peroxidase and, if using avidin–biotin detection, endogenous biotin as possible sources of signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A00336 paraffin-section caption does not state a fixative (caption: fixative unreported). The caption uses Tris-EDTA retrieval at pH 9.0, but does not establish that retrieval is required (caption: Tris-EDTA, pH 9.0); the supplied evidence does not establish whether frozen sections or IF are easier. Kidney endogenous biotin can complicate avidin–biotin detection (standard IHC practice).
HPA tissue IHC evidence for MTHFR
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bronchus | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Pituitary gland | Cells in anterior | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MTHFR IHC Tips
Troubleshoot MTHFR staining in paraffin sections by checking retrieval, cell type, staining pattern, and controls alongside the catalog antibody’s tissue image.
How should I retrieve MTHFR in paraffin sections when staining is weak?
Start with heat-mediated Tris-EDTA at pH 9.0 for MTHFR antigen retrieval (datasheet A00336). The catalog antibody’s paraffin-section image used that buffer and pH, with primary antibody at 1:200 overnight at 4°C (caption A00336). If staining is weak, compare a fresh retrieval run with an established positive control, such as kidney tubules or duodenal glandular cells, while keeping detection conditions constant (HPA: High in these cells; standard IHC practice). Record heating and cooling conditions for each run, because inconsistent heat exposure can change chromogenic signal and tissue morphology independently of antibody performance (standard IHC practice).
Can I infer the right fixative for MTHFR from the available paraffin image?
No target-specific fixation sensitivity is established here: the catalog image identifies a paraffin-embedded specimen but does not state its fixative (caption A00336). Record the fixative and fixation duration for each specimen, then compare sections processed under matched conditions before attributing a weak signal to MTHFR biology (standard IHC practice). Keep the documented Tris-EDTA retrieval at pH 9.0 and primary incubation at 1:200 overnight at 4°C consistent during that comparison (caption A00336). Include a known staining-positive cell population on each run, and assess tissue preservation and background alongside signal intensity (HPA: High in kidney tubules; standard IHC practice).
How should I interpret cytoplasmic versus junctional MTHFR staining?
Assess the pattern within identified cells before scoring intensity: tissue IHC reports cytoplasmic expression across several tissues, while subcellular IF reports mainly cell junctions with additional centrosome and basal-body localisation (HPA: tissue profile; HPA: subcellular summary). These observations come from different assay contexts, so a junctional rim in paraffin sections needs comparison with the cytoplasmic signal, cell morphology, and controls rather than automatic acceptance (HPA: tissue profile; HPA: subcellular summary; standard IHC practice). MTHFR has no annotated transmembrane segment, which does not by itself identify the epitope or prove a staining compartment (UniProt P42898 topology). Document compartment-specific staining separately in the same cell population when patterns disagree (standard IHC practice).
Could isoforms or phosphorylation explain discordant MTHFR staining?
MTHFR has 2 annotated isoforms and multiple modified residues, including phosphoserines at positions 9, 10, 18, 20, and 21 (UniProt P42898 isoforms; modified residues). The supplied material does not map the catalog antibody’s epitope, so isoform recognition and sensitivity to those modifications cannot be assigned (caption A00336; UniProt P42898). When comparing specimens, keep retrieval at Tris-EDTA pH 9.0 and primary dilution at 1:200, then record whether staining changes by cell type or compartment (caption A00336; standard IHC practice). Resolve an epitope-specific explanation only with epitope information or an independent specificity control, rather than staining appearance alone (standard IHC practice).
How can IF help resolve an ambiguous MTHFR IHC pattern?
Use IF as a separate localisation check, since subcellular images report MTHFR mainly at cell junctions and additionally at centrosomes and basal bodies (HPA: subcellular summary). Multiplex with a marker for the cell population under study, such as a tubular epithelial marker when examining kidney tubules, so MTHFR signal can be assigned to the expected cells (HPA: High in kidney tubule cells; standard IF practice). Choose a fluorophore channel away from prominent tissue autofluorescence and include unstained and single-label controls before judging overlap (standard IF practice). Because the epitope is unspecified and MTHFR has no annotated transmembrane segment, compare permeabilised and non-permeabilised preparations before interpreting intracellular access or apparent surface signal (UniProt P42898 topology; standard IF practice).
What should I check when MTHFR chromogenic staining looks diffuse or excessive?
Run a no-primary control to identify signal from the chromogenic detection system, and inspect sections for pigment, damaged edges, and uneven reagent coverage (standard IHC practice). Include a peroxidase block before peroxidase-based detection, and evaluate DAB development as a general workflow variable rather than evidence specific to MTHFR (standard IHC practice). Compare the affected cells with documented contrasts: kidney tubule cells stain High, whereas adipocytes in adipose tissue are Not detected in the HPA tissue dataset (HPA: kidney; HPA: adipose tissue). If background persists, titrate the primary around the caption’s 1:200 condition and keep retrieval and development consistent across comparison sections (caption A00336; standard IHC practice).
How should I score MTHFR staining across paraffin-section samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and staining compartment before scoring, because HPA tissue IHC describes cytoplasmic staining whereas subcellular IF highlights cell junctions (HPA: tissue profile; HPA: subcellular summary). For cell-based comparisons, report percent positive cells and an intensity-weighted H-score on its conventional 0–300 scale; record cytoplasmic and junctional patterns separately if both occur (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable, annotated tissue rather than total slide area (standard IHC practice). Normalise measurements to the same cell type, sampled area, staining run, and exposure or development conditions, using a shared positive control to monitor run variation (standard IHC practice).
What distinguishes credible MTHFR positivity from a staining artefact?
Credible staining should be reproducible in an identified cell population and broadly compatible with the tissue-IHC cytoplasmic profile, while junctional signal merits separate assessment because it is prominent in subcellular IF (HPA: tissue profile; HPA: subcellular summary; standard IHC practice). Kidney tubule cells provide a documented High-staining comparison, and adipocytes in adipose tissue provide a Not detected comparison within the HPA dataset (HPA: kidney; HPA: adipose tissue). Treat signal confined to section edges, necrotic regions, or the no-primary control as suspect, and check endogenous peroxidase activity when using peroxidase-based detection (standard IHC practice). Interpret modest differences cautiously because HPA rates its tissue staining Enhanced but reports medium consistency with RNA expression (HPA: reliability description).
Boster reagents
Best MTHFR / Methylenetetrahydrofolate reductase (NADPH) IHC Antibodies
A00336 and M00336 have real IHC images of paraffin-embedded human tissues (catalog IHC captions). Listed species reactivity covers human, monkey, mouse, and rat; IF is listed for M00336 (catalog applications and reactivity).
Real IHC data
Anti-MTHFR Antibody
Cat # A00336
Real IHC data
Anti-MTHFR Monoclonal Antibody
Cat # M00336
A00336 has an IHC image of paraffin-embedded human oophoroma and lists human, monkey, and mouse reactivity (A00336 IHC caption; catalog reactivity). M00336 has a DAB IHC image of paraffin-embedded human lung cancer tissue, lists human and rat reactivity, and includes IF among its applications (M00336 IHC caption; catalog applications and reactivity).
Which to pick: For paraffin-section IHC, choose A00336 for its rabbit host and human, monkey, and mouse reactivity, with human oophoroma IHC evidence; choose M00336 for a mouse monoclonal (clone 5D3) with human and rat reactivity and human lung cancer IHC evidence (catalog host, clone, and reactivity; each SKU’s IHC caption). For IF planning, choose M00336 because IF is listed at 1:50; ICC validation is unreported (M00336 catalog applications and dilution). The fixative is unreported for both IHC images (A00336 and M00336 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P42898 (MTHR_HUMAN, Methylenetetrahydrofolate reductase (NADPH)).
- Human Protein Atlas. MTHFR tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. MTHFR subcellular location (ICC-IF): Mainly localized to the cell junctions. In addition localized to the centrosome and basal body..
- Human Protein Atlas. MTHFR antibody validation summary (3 antibodies).
- MTHFR act as a potential cancer biomarker in immune checkpoints blockades, heterogeneity, tumor microenvironment and immune infiltration. Discover oncology 2023 — PMC10290629.
- LncRNA HOTAIR-mediated MTHFR methylation inhibits 5-fluorouracil sensitivity in esophageal cancer cells. Journal of experimental & clinical cancer research : CR 2020 — PMC7353690.
- Mthfr as a modifier of the retinal phenotype of Crb1(rd8/rd8) mice. Experimental eye research 2016 — PMC4842092.
- Modulation of blood pressure regulatory genes in the Agtrap-Plod1 locus associated with a deletion in Clcn6. Physiological reports 2022 — PMC9353118.
- PubMed PMID:9680386 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MTHFR (Methylenetetrahydrofolate reductase (NADPH)). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A00336). Validated IHC antibody: A00336 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Ciliated cells (cilia axoneme)); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human oophoroma tissue; fixative not specified (datasheet A00336); 2. Fixation: Image fixative and duration unreported (datasheet A00336); 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: Tris-EDTA pH 9.0 (datasheet A00336); 20 min, 95–100 °C (standard); 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-MTHFR, 1:50 recommended; image 1:200 (datasheet A00336); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has only medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Staining-linked regulation is unreported (UniProt). Evidence: UniProt P42898, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cell junctions. In addition localized to the…, Datasheet A00336, PMC10290629, PMC7353690. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MTHFR/immunohistochemistry-ihc-guide.