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SHMT2 / Serine hydroxymethyltransferase, mitochondrial · IHC design guide
Design Immunohistochemistry for SHMT2
Plan SHMT2 chromogenic IHC in paraffin sections using hepatocytes or kidney proximal tubules as high-staining reference cells (HPA tissue IHC). This guide covers fixation, controls and interpretation of the cytoplasmic tissue pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SHMT2 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 staining in most tissues, including hepatocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03233-1) | |
| Positive control | Appendix+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Cytoplasmic IHC cannot resolve mitochondrial location (HPA tissue IHC; UniProt) | |
| Regulation | Stimulus-linked regulation not specified (UniProt) | |
| Isoform / epitope | 3 isoforms; mature chain 30–504; epitope coverage unknown (UniProt) |
Section 1
Recommended SHMT2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A03233-1) is accompanied by three published SHMT2 tissue-staining protocols (PMC8052717; PMC7581701; PMC5538688).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A03233-1) |
| Fixation | Image fixative and duration unreported (datasheet A03233-1); 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 A03233-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03233-1) |
| Primary antibody | Rabbit anti-SHMT2, 2-5 μg/ml (datasheet A03233-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03233-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03233-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SHMT2-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 (datasheet A03233-1). For oral squamous cell carcinoma microarrays, a published alternative is microwave retrieval in citrate at pH 6.0 (PMC7581701).
Section 2
What Is the Expected SHMT2 Staining Pattern?
SHMT2 should show mainly cytoplasmic staining in IHC, consistent with its predominant mitochondrial localization (HPA: cytoplasmic expression in most tissues; UniProt P34897: mainly mitochondrial). High staining is reported in hepatocytes, kidney proximal tubules, and several glandular or lymphoid cell populations (HPA: tissue IHC). Its mitochondrial matrix and inner membrane annotations do not imply cell-surface staining; no transmembrane segment is annotated (UniProt P34897 topology).
| Cytoplasmic staining in hepatocytes, kidney proximal tubules, or intestinal glandular cells. | This fits reported high IHC staining in those cell populations (HPA: High in hepatocytes, proximal tubules, and small-intestinal glandular cells). A finely granular appearance would be consistent with mitochondrial localization, but chromogenic IHC alone cannot establish organelle identity (UniProt P34897: mainly mitochondrial; general IHC practice). |
| Predominantly membrane-rim or exclusively nuclear staining, with little cytoplasmic signal. | Recheck staining specificity and slide interpretation: the dominant pattern conflicts with HPA's cytoplasmic tissue profile and UniProt's mainly mitochondrial localization (HPA: tissue IHC; UniProt P34897). Nuclear localization is also annotated, so limited nuclear signal alone is not proof of an artefact (UniProt P34897). |
| Strong staining in adipocytes, cardiomyocytes, or skeletal myocytes. | These cell populations are reported as not detected by HPA tissue IHC (HPA: Not detected in adipocytes, cardiomyocytes, and skeletal myocytes). Treat a strong result as discordant and check cell identification, cross-reactivity, and chromogen generated by endogenous activity before assigning it to SHMT2 (general IHC practice). |
| Diffuse stain covers tissue and blank spaces, obscuring cell boundaries. | A widespread haze does not resolve the reported cell-type pattern (HPA: tissue IHC). Check reagent background, incomplete blocking or washing, and overdevelopment using appropriate detection controls (general IHC practice). This appearance alone cannot establish SHMT2 distribution (general IHC practice). |
| No staining in hepatocytes or kidney proximal tubules. | Both are reported as High in HPA tissue IHC, making them useful positive reference populations (HPA: High in hepatocytes and proximal tubules). First check tissue preservation, retrieval, antibody and detection performance, and a concurrent positive control; a single negative section does not overturn the reference pattern (general IHC practice). |
💡Expected SHMT2 appearanceCall positive when the expected cells show clear cytoplasmic staining, with high signal in HPA-listed populations such as hepatocytes or proximal tubules; diffuse haze or dominant staining of HPA-listed undetected cells is suspect (HPA: tissue IHC; general IHC practice).
| Cell population and reference tissue | HPA reports High staining in liver hepatocytes, bronchial ciliated cell bodies, kidney proximal tubules, and selected glandular or lymphoid cells; it reports several stromal and muscle populations as Not detected (HPA: tissue IHC). Compare the same cell population, since a tissue can contain multiple cell types (general IHC practice). |
| Compartment and topology | The mainly mitochondrial protein is annotated in the matrix, nucleoid, inner membrane, cytoplasm, and nucleus, with no transmembrane segment (UniProt P34897). HPA tissue IHC summarizes its pattern as cytoplasmic; routine chromogenic sections cannot distinguish those mitochondrial subcompartments reliably (HPA: tissue IHC; general IHC practice). |
| Antibody validation and scope | HPA lists two rabbit polyclonal antibodies, HPA020543 and HPA020549, with Enhanced IHC validation (HPA: antibody validation). The tissue profile itself has Enhanced reliability with medium consistency between antibody staining and RNA expression; use a matched control when interpreting a surprising sample (HPA: tissue IHC; general IHC practice). |
| Processing and isoforms | UniProt annotates a processed chain spanning residues 30–504 and three isoforms (UniProt P34897). The supplied record gives no antibody epitope, so recognition of every isoform or processed form cannot be inferred; consult the antibody-specific documentation before attributing a missing signal to processing (UniProt P34897; general IHC practice). |
| IF/ICC Q&A: where should fluorescence localize? | Mainly to mitochondria, with an additional approved microtubule location in HPA cell images (HPA: subcellular ICC-IF). UniProt also annotates cytoplasmic and nuclear localization (UniProt P34897). These IF observations help interpret compartment differences; they do not specify an IHC staining protocol (HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known positive tissue has no visible signal. | The staining run or tissue section may have failed; HPA reports High signal in hepatocytes and kidney proximal tubules (HPA: tissue IHC; general IHC practice). | Check a concurrent positive section, reagent integrity, retrieval conditions, detection steps, and counterstain strength; adjust only against controls (general IHC practice). |
| Signal is weak in the expected cytoplasm. | A weak assay or variable specimen may obscure the reported High cell populations; HPA's tissue profile has medium staining-to-RNA consistency (HPA: tissue IHC; general IHC practice). | Compare the same cell type on a reference section and review retrieval and antibody titration with the IHC-validated antibody (general IHC practice). |
| Diffuse brown background overwhelms cells. | Nonspecific reagent binding, endogenous detection activity, or excess chromogen development can reduce contrast (general IHC practice). | Use an appropriate negative or detection-only control; review blocking, washing, and development time before interpreting cellular signal (general IHC practice). |
| HPA-listed undetected cells stain strongly. | Cross-reactivity, endogenous detection activity, or misidentified cells are possible; HPA reports Not detected in adipocytes and cardiomyocytes (HPA: tissue IHC; general IHC practice). | Confirm the cell type and compare with a positive reference and detection control; avoid calling SHMT2 from intensity alone (general IHC practice). |
| Only nuclei or cell borders stain. | A dominant nuclear or membrane-rim pattern conflicts with the mainly cytoplasmic tissue profile, though UniProt permits some nuclear localization (HPA: tissue IHC; UniProt P34897). | Review morphology and controls, then compare with the expected cytoplasmic signal; do not discard limited nuclear signal solely on location (general IHC practice; UniProt P34897). |
| Two specimens show different staining intensity. | Different cell populations or specimen quality may contribute; HPA reports High, Low, and Not detected populations rather than one universal intensity (HPA: tissue IHC; general IHC practice). | Score the same cell population across specimens with a shared positive control and consistent detection settings; record compartment and background separately (general IHC practice). |
Experiment evidence
IHC & IF images showing SHMT2 staining
A03233-1 · IHCIHC analysis of SHMT2 using anti-SHMT2 antibody (A03233-1). SHMT2 was detected in a paraffin-embedded section of human placenta tissue. Heat
A03233-1 · IHCIHC analysis of SHMT2 using anti-SHMT2 antibody (A03233-1). SHMT2 was detected in a paraffin-embedded section of human bladder cancer tissue
A03233-1 · IFIF analysis of SHMT2 using anti-SHMT2 antibody (A03233-1). SHMT2 was detected in an immunocytochemical section of U2OS cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000182199-SHMT2/tissue/cerebral+cortex#imid_5612260
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000182199-SHMT2/tissue/colon#imid_5612178
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000182199-SHMT2/tissue/liver#imid_5612187
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000182199-SHMT2/tissue/kidney#imid_5612180
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020543; green = SHMT2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020543; green = SHMT2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SHMT2 IHC & IF
🧪Run kidney first: proximal tubule cell bodies should stain (HPA: High in kidney proximal tubules). Use adipose tissue as a negative tissue: adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the kidney slide, unstained cells outside the proximal tubules can show background, but they are not established SHMT2-negative cells (HPA: kidney row identifies proximal tubules only).
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SHMT2 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a concentration-matched rabbit IgG isotype control, and an SHMT2-knockout biological negative (caption: rabbit primary antibody; standard IHC practice). For chromogenic kidney IHC, quench endogenous peroxidase and check for endogenous biotin signal if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03233-1 tissue-IHC caption does not state the fixative (selected-SKU caption). The caption reports heat retrieval in EDTA at pH 8.0 for a paraffin-embedded placenta section; whether SHMT2 staining depends on that retrieval condition is unreported (selected-SKU caption). Frozen sections and IF are not established as easier by the supplied evidence; kidney proximal tubules warrant attention to endogenous biotin during chromogenic detection (HPA: High in kidney proximal tubules; standard IHC practice).
HPA tissue IHC evidence for SHMT2
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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (cell body) | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SHMT2 IHC Tips
Troubleshoot SHMT2 staining in paraffin sections by checking retrieval, cellular distribution, controls, and scoring before interpreting signal intensity.
Which retrieval conditions should I try first for weak SHMT2 staining?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A03233-1). The selected paraffin-section example used that retrieval before an overnight incubation with 2 μg/ml antibody at 4°C (datasheet A03233-1). If staining is weak, compare a shorter and longer heating interval on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Inspect tissue integrity and nonspecific staining alongside the expected cellular signal, since excessive heating can damage morphology (standard IHC practice). Only then test another retrieval buffer as a fallback, recording its pH and heating conditions separately (standard IHC practice).
Could fixation explain weak or uneven SHMT2 staining?
Target-specific sensitivity of SHMT2 staining to fixative type or duration is unknown from the supplied evidence (selected A03233-1 caption: fixative not stated). The selected image documents a paraffin-embedded placenta section, but its caption does not identify how that tissue was fixed (datasheet A03233-1). Record the fixative, fixation duration, processing history, section thickness, and age of each slide before comparing staining across specimens (standard IHC practice). For a suspected processing effect, stain matched sections in the same run using the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration (datasheet A03233-1; standard IHC practice). Judge differences against preserved morphology and an appropriate positive control (standard IHC practice).
How should punctate and diffuse SHMT2 staining be assessed?
Assess SHMT2 primarily within the cytoplasmic compartment, where mitochondrial signal may appear granular at the resolution of chromogenic IHC (UniProt P34897 localisation; standard IHC practice). The protein mainly localizes to mitochondria and is also reported in cytoplasm and nucleus as part of the BRISC complex (UniProt P34897 localisation). HPA describes cytoplasmic expression in most tissues, while its subcellular data support mitochondria and additionally report microtubules (HPA tissue IHC; HPA subcellular). Compare the pattern with cell morphology and a matched negative control before assigning a specific organelle from DAB alone (standard IHC practice). Record diffuse cytoplasmic and nuclear staining separately rather than treating either pattern as proof of mislocalisation (UniProt P34897 localisation; standard IHC practice).
Can SHMT2 processing or isoforms change the staining pattern?
SHMT2 has 3 annotated isoforms and a processed chain spanning residues 30–504, but the supplied antibody caption does not identify its epitope (UniProt P34897 isoforms and processing; datasheet A03233-1). An epitope in a region absent from an isoform or altered by processing could affect recognition, so check the antibody's documented immunogen before assigning an isoform-specific interpretation (UniProt P34897 isoforms and processing; standard IHC practice). The record also lists modified residues, including acetyllysines, without establishing an effect on this antibody's staining (UniProt P34897 modified residues; datasheet A03233-1). Compare any unexplained pattern with an independently validated antibody against a distinct epitope, using matched sections and controls (standard IHC practice).
How can IF help assess the cellular source of SHMT2 staining?
For a separate IF experiment, pair SHMT2 with a marker identifying the expected cell type in the specimen and assess overlap at single-cell resolution (standard IF practice). Select fluorophores whose emission can be distinguished from the specimen's autofluorescence, and include unstained and single-label controls (standard IF practice). SHMT2 is mainly mitochondrial and has no annotated transmembrane segment, so permeabilise sufficiently to expose an intracellular epitope after fixation while preserving mitochondrial structure (UniProt P34897 topology and localisation; standard IF practice). Compare the resulting distribution with HPA's supported mitochondrial localisation, but do not equate every cytoplasmic signal with mitochondria (HPA subcellular; standard IF practice). Optimise IF fixation and permeabilisation independently because the selected IHC caption provides no IF fixation conditions (datasheet A03233-1).
What should I change when DAB background obscures SHMT2 staining?
First compare the stained section with a no-primary control and inspect whether colour follows tissue edges, damaged areas, or the expected cellular pattern (standard IHC practice). The selected paraffin-section example used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (datasheet A03233-1). Confirm a peroxidase block and adequate washes before DAB development, since residual enzyme activity and retained detection reagents can produce background (standard chromogenic IHC practice). Titrate primary antibody around the documented concentration and shorten chromogen development if the control also darkens (datasheet A03233-1; standard IHC practice). Keep exposure and processing comparable across test sections (standard IHC practice).
How should SHMT2 staining be scored across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then report the percentage of positive cells together with intensity or an H-score (standard IHC practice). If cell density varies, report positive cells per mm² and normalise measurements to the relevant viable tissue area or eligible cell count (standard IHC practice). Score cytoplasmic and nuclear signal separately because both locations are reported for SHMT2, while mitochondria are its main location (UniProt P34897 localisation). Exclude folds, tissue edges, necrotic regions, and obvious pigment from the analysis mask (standard IHC practice). Apply the same threshold, retrieval, detection, and imaging settings to all compared slides, with blinded scoring where feasible (standard IHC practice).
What distinguishes credible SHMT2 staining from an IHC artefact?
A credible result shows reproducible cellular staining with appropriate control behaviour and a pattern compatible with SHMT2's predominantly mitochondrial location (UniProt P34897 localisation; standard IHC practice). HPA reports high staining in hepatocytes and kidney proximal tubules, whereas adipocytes and cardiomyocytes were not detected in its tissue survey; use these observations as context, not absolute rules for every preparation (HPA tissue IHC). Diffuse signal confined to section edges, necrosis, or a no-primary control suggests an artefact requiring investigation (standard IHC practice). Check endogenous peroxidase activity when DAB appears without primary antibody (standard chromogenic IHC practice). Interpret nuclear signal cautiously because a nuclear pool is reported, but DAB alone cannot establish its molecular role (UniProt P34897 localisation; standard IHC practice).
Boster reagents
Best SHMT2 / Serine hydroxymethyltransferase, mitochondrial IHC Antibodies
A03233-1 has IHC images from human paraffin sections and IF images from U2OS cells and human paraffin sections (A03233-1 image captions). Human, mouse and rat reactivity is listed (catalog: reactivity).
Real IHC data
Anti-SHMT2 Antibody ®
Cat # A03233-1
A03233-1 is shown in chromogenic IHC on human placenta, bladder cancer, lung cancer and liver cancer paraffin sections (A03233-1 IHC captions). The same SKU is shown in IF on U2OS cells and human colon and thyroid cancer paraffin sections (A03233-1 IF captions).
Which to pick: For tissue IHC, choose A03233-1 at the listed 2–5 μg/ml range (catalog: IHC dilution); its own IHC captions show EDTA retrieval and DAB detection in paraffin sections, but do not report the fixative (A03233-1 IHC captions). For IF/ICC, A03233-1 lists both applications at 5 μg/ml and has corresponding cell and tissue IF images (catalog: applications and IF dilution; A03233-1 IF captions). For mouse or rat work, A03233-1 lists both species as reactive, while its shown IHC images use human tissue (catalog: reactivity; A03233-1 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P34897 (GLYM_HUMAN, Serine hydroxymethyltransferase, mitochondrial).
- Human Protein Atlas. SHMT2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. SHMT2 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the microtubules..
- Human Protein Atlas. SHMT2 antibody validation summary (2 antibodies).
- Silencing SHMT2 inhibits the progression of tongue squamous cell carcinoma through cell cycle regulation. Cancer cell international 2021 — PMC8052717.
- SHMT2 Drives the Progression of Colorectal Cancer by Regulating UHRF1 Expression. Canadian journal of gastroenterology & hepatology 2022 — PMC8863481.
- Increased Expression of SHMT2 Is Associated With Poor Prognosis and Advanced Pathological Grade in Oral Squamous Cell Carcinoma. Frontiers in oncology 2020 — PMC7581701.
- Overexpression of mitochondrial serine hydroxyl-methyltransferase 2 is associated with poor prognosis and promotes cell proliferation and invasion in gliomas. OncoTargets and therapy 2017 — PMC5538688.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16541075 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SHMT2 (Serine hydroxymethyltransferase, mitochondrial). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03233-1). Validated IHC antibody: A03233-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A03233-1); 2. Fixation: Image fixative and duration unreported (datasheet A03233-1); 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 A03233-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03233-1); 8. Primary antibody: Rabbit anti-SHMT2, 2-5 μg/ml (datasheet A03233-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03233-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Cytoplasmic IHC cannot resolve mitochondrial location (HPA tissue IHC; UniProt); Isoform / epitope: 3 isoforms; mature chain 30–504; epitope coverage unknown (UniProt); Regulation: Stimulus-linked regulation not specified (UniProt). Evidence: UniProt P34897, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A03233-1, PMC8052717, PMC7581701. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SHMT2/immunohistochemistry-ihc-guide.