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PHGDH / D-3-phosphoglycerate dehydrogenase · IHC design guide
Design Immunohistochemistry for PHGDH
Plan PHGDH staining in paraffin sections using its reported cytoplasmic and occasional nuclear tissue pattern (HPA tissue IHC). The guide covers fixation, antigen retrieval, antibody incubation, chromogenic detection and interpretation of the tissue evidence.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PHGDH 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 | General cytoplasmic staining; nuclear in several tissues (HPA tissue IHC) | |
| Staining pattern | Glandular and other cells: cytoplasmic, sometimes nuclear (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03355-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03355-1) | |
| Caveat | Antibody staining and RNA show only medium consistency (HPA tissue IHC) | |
| Regulation | Tissue-level regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; the mature chain spans residues 2–533 (UniProt) |
Section 1
Recommended PHGDH IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: A03355-1). Published PHGDH IHC protocols below provide additional sample preparation, detection, and scoring context (PMC8871196; PMC4907894; PMC3107855).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A03355-1) |
| Fixation | Image fixative and duration unreported (datasheet A03355-1); 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 A03355-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03355-1) |
| Primary antibody | Rabbit anti-PHGDH, 2-5 μg/ml (datasheet A03355-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03355-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03355-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PHGDH-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: A03355-1). For the published endometrial protocol, use its stated citrate retrieval (PMC3107855 methods).
Section 2
What Is the Expected PHGDH Staining Pattern?
PHGDH staining in tissue is generally cytoplasmic, with nuclear staining in several tissues (HPA tissue IHC). Expect strong staining in selected glandular, basal, glial and endothelial cells, depending on the tissue (HPA tissue IHC). HPA rates the tissue profile Enhanced, while noting medium agreement with RNA data and pending external verification (HPA tissue IHC). PHGDH has no annotated transmembrane segment (UniProt O43175 topology).
| Clear cytoplasmic staining in appendix or breast glandular cells, with possible nuclear staining (HPA tissue IHC). | This matches PHGDH’s reported tissue pattern; both cell identity and compartment matter when judging a positive slide (HPA tissue IHC). Compare intensity with a low-staining cell population on the same run where available, while allowing for tissue-to-tissue variation (HPA tissue IHC; standard IHC practice). |
| Signal appears mainly in extracellular material, section edges or debris, without the expected cellular pattern. | This does not match the reported general cytoplasmic and occasional nuclear tissue pattern (HPA tissue IHC). Check morphology and controls before scoring it as PHGDH; edge and debris staining can reflect staining artefact (standard IHC practice). |
| Strong staining appears in an unexpected cell population while the expected cells stain weakly. | Question specificity when staining conflicts with the cell-level reference pattern (HPA tissue IHC). Primary-antibody cross-reactivity is possible; chromogen in a no-primary control instead points toward endogenous detection activity or another detection-stage background source (standard IHC practice). |
| A uniform haze covers cells and surrounding tissue, obscuring cellular boundaries. | Diffuse background prevents a reliable compartment or cell-type call (standard IHC practice). Inspect a no-primary control and review blocking, washes and detection conditions before comparing the slide with HPA’s cellular pattern (HPA tissue IHC; standard IHC practice). |
| No cellular signal appears in a section containing appendix or breast glandular cells. | Those cells are reported as High in HPA tissue IHC, so an entirely blank positive control calls the run into question (HPA tissue IHC). Check tissue integrity, antigen retrieval, antibody use and detection controls before treating the result as biological absence (standard IHC practice). |
💡Expected PHGDH appearanceA convincing positive shows distinct, often strong cytoplasmic staining in an HPA High cell population, with nuclear staining possible in some tissues; extracellular or uniform hazy chromogen is suspect (HPA tissue IHC; standard IHC practice).
| Cell-level tissue reference | HPA reports High staining in appendix and breast glandular cells, bronchus basal cells, caudate glial cells and cerebral-cortex endothelial cells (HPA tissue IHC). It reports Low staining in lung alveolar cells and heart cardiomyocytes, among others (HPA tissue IHC). Choose comparisons by cell type, since a whole-section average can hide the relevant population (standard IHC practice). |
| Compartment and topology | Tissue IHC reports general cytoplasmic staining and nuclear staining in several tissues (HPA tissue IHC). PHGDH has no annotated transmembrane segment (UniProt O43175 topology). A membrane-like outline alone therefore needs cellular and control-based review; it should not override the tissue IHC reference (HPA tissue IHC; standard IHC practice). |
| Strength of the reference evidence | The tissue profile is rated Enhanced but has medium consistency with RNA expression and awaits external verification (HPA tissue IHC). Among listed antibodies, CAB003681 has Enhanced IHC validation; HPA021241, HPA024031 and CAB068216 are Approved for IHC (HPA antibodies). Interpret disagreement cautiously and record which antibody produced the slide (standard IHC practice). |
| Fixation and retrieval evidence | Target-specific fixation sensitivity and an optimal retrieval condition are unreported in the supplied UniProt and HPA records. For an IHC-P run, follow the IHC-validated antibody’s documented starting conditions when available, then assess the positive tissue and controls together (standard IHC practice). |
| IF/ICC Q: Where might PHGDH fluorescence appear? | A: HPA ICC-IF reports approved nucleoplasm, plasma-membrane and cytosol localization, plus mitotic-spindle and primary-cilium localization (HPA subcellular ICC-IF). Those cell-image observations add context; they do not establish that every compartment should be visible in a paraffin tissue IHC section (HPA tissue IHC; HPA subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected High cells are blank, and the run’s positive control is also blank. | A shared processing or detection failure is possible; appendix or breast glandular cells are reported High (HPA tissue IHC; standard IHC practice). | Verify the antibody, retrieval, detection reagents and counterstained tissue integrity against the documented IHC-P workflow; rerun a suitable positive control before interpreting absence (standard IHC practice). |
| Expected cells are blank, but the run’s positive control stains appropriately. | The sampled cells may differ from the reference population, or the section may lack interpretable target cells (HPA tissue IHC; standard IHC practice). | Recheck cell identity and morphology; compare an HPA High population in a matched run before assigning a negative score (HPA tissue IHC; standard IHC practice). |
| Chromogen persists in the no-primary control. | Endogenous detection activity or detection-stage background can produce signal without primary antibody (standard IHC practice). | Review the control’s distribution and apply the blocking or detection controls appropriate to the chosen chromogen system; score PHGDH only after background is controlled (standard IHC practice). |
| Unexpected cells stain strongly, while the no-primary control is clean. | Primary-antibody cross-reactivity is possible, although PHGDH intensity varies by cell type and tissue (HPA tissue IHC; standard IHC practice). | Compare the cell identity and compartment with HPA tissue IHC; where feasible, check the pattern with a separately validated PHGDH antibody (HPA antibodies; standard IHC practice). |
| A diffuse haze obscures the expected cellular pattern. | Background from the staining workflow can mask interpretable cytoplasmic staining (HPA tissue IHC; standard IHC practice). | Inspect the no-primary control, then review blocking, antibody dilution, washing and chromogen development using the documented workflow (standard IHC practice). |
| Staining is confined to section edges, folds or damaged areas. | Local section artefact can mimic a positive signal without reproducing the reported cell-level pattern (HPA tissue IHC; standard IHC practice). | Evaluate intact tissue away from the affected area, compare the control section and repeat staining on a usable section if the relevant cells cannot be scored (standard IHC practice). |
Experiment evidence
IHC & IF images showing PHGDH staining
A03355-1 · IHCIHC analysis of PHGDH using anti-PHGDH antibody (A03355-1). PHGDH was detected in a paraffin-embedded section of human breast cancer tissue.
A03355-1 · IHCIHC analysis of PHGDH using anti-PHGDH antibody (A03355-1). PHGDH was detected in a paraffin-embedded section of mouse brain tissue. Heat me
A03355-1 · IFIF analysis of PHGDH using anti-PHGDH antibody (A03355-1). PHGDH was detected in an immunocytochemical section of U2OS cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000092621-PHGDH/tissue/cerebral+cortex#imid_18844115
HPA · Salivary glandHuman Protein Atlas IHC, Salivary gland. Source: https://www.proteinatlas.org/ENSG00000092621-PHGDH/tissue/salivary+gland#imid_18844063
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000092621-PHGDH/tissue/colon#imid_18844041
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000092621-PHGDH/tissue/liver#imid_18844034
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA021241; green = PHGDH, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA021241; green = PHGDH, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PHGDH IHC & IF
🧪Run breast tissue first and assess staining in glandular cells, where HPA reports High PHGDH expression (HPA: breast glandular cells, High). HPA detects PHGDH in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat any unstained non-glandular cells on the positive slide as a background comparison only, not as a validated PHGDH-negative population (HPA: no negative tissue rows).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: PHGDH 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 PHGDH in A-431, U-251MG, U2OS, MCF-7, PC-3, KOLF2.1J, NIH 3T3, with annotated localisation: Nucleoplasm (approved), Plasma membrane (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control appropriate to the primary, and a PHGDH-knockout specimen or peptide-blocked primary as a biological specificity control (selected A03355-1 caption: rabbit primary). Quench endogenous peroxidase in breast sections before HRP–DAB detection; assess endogenous biotin if using a biotin-based detection system (selected A03355-1 caption: HRP–DAB detection).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected paraffin-section caption does not state the fixative (selected A03355-1 caption: fixative unreported). The reported IHC result used heat-mediated retrieval in EDTA, pH 8.0, so use that as the starting retrieval condition; whether retrieval is essential is unreported (selected A03355-1 caption: EDTA retrieval). The supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC, or identify a breast-specific artefact; check background in glandular and adjacent tissue compartments (HPA: breast glandular cells, High; selected A03355-1 caption: paraffin-section IHC).
HPA tissue IHC evidence for PHGDH
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Basal cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: PHGDH is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced PHGDH IHC Tips
Troubleshoot PHGDH staining in paraffin sections by checking retrieval, controls, cellular localization and scoring before comparing results across specimens.
What retrieval should I try first for weak PHGDH staining?
Start with heat-mediated EDTA at pH 8.0 for paraffin-section PHGDH IHC (datasheet A03355-1). The selected image uses this retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C; reproduce those conditions when assessing a weak result (datasheet A03355-1). If staining remains weak, vary heating duration and cooling consistently across matched sections while retaining EDTA at pH 8.0 as the starting condition (standard IHC practice). Compare a no-primary section and breast glandular cells as a documented high-staining reference to help separate retrieval failure from background (standard IHC practice; HPA: High in breast glandular cells).
Could fixation explain inconsistent PHGDH staining between specimens?
PHGDH-specific sensitivity to fixation is unknown because the selected paraffin-section caption does not state a fixative (datasheet A03355-1). Record each specimen’s fixative, fixation duration and processing history, then compare matched sections using EDTA at pH 8.0 and 2 μg/ml primary antibody under consistent conditions (standard IHC practice; datasheet A03355-1). If staining differs, inspect morphology and a no-primary control before attributing the difference to antigen loss; fixation can affect epitope accessibility and tissue background (standard IHC practice). Neither the recorded PHGDH modifications nor its HPA tissue pattern establishes a PHGDH-specific fixation tolerance (UniProt O43175; HPA: tissue IHC).
Should PHGDH appear in the cytoplasm, nucleus or membrane?
Expect predominantly cytoplasmic staining in tissue, with nuclear staining in some tissues (HPA: general cytoplasmic expression with nuclear expression in several tissues). HPA cell imaging also places PHGDH in the nucleoplasm, plasma membrane and cytosol, with additional mitotic spindle and primary cilium localization (HPA: subcellular). UniProt annotates no transmembrane segment or subcellular location, so corroborate a sharply membranous or exclusively nuclear DAB pattern before treating it as specific (UniProt O43175 topology and localization; standard IHC practice). Score compartments separately using cellular morphology and documented breast glandular staining as reference points (HPA: High in breast glandular cells; standard IHC practice).
Could isoforms or epitope placement explain discordant staining?
No alternative PHGDH isoforms or domains are annotated, and the recorded processed chain spans residues 2–533 (UniProt O43175). The supplied catalog antibody information does not map its epitope, so staining cannot be assigned to a particular region or modification from these data (datasheet A03355-1; UniProt O43175). Recorded modifications include phosphoserine at 14, acetyllysine at 21 and phosphothreonine at 78; modifications can affect epitope recognition in principle, but no such effect is established here (UniProt O43175; standard IHC practice). For discordant specimens, compare a second antibody with a mapped, nonoverlapping epitope on matched sections and assess agreement by cell type and compartment (standard IHC practice).
How should I adapt the IHC finding to multiplex IF?
Optimize IF independently: the selected antibody evidence describes paraffin-section chromogenic IHC and supplies no IF protocol (datasheet A03355-1). Pair PHGDH with a validated cell-type marker, such as a glandular-cell marker when examining breast, and check single-color controls for spectral overlap (HPA: High in breast glandular cells; standard IF practice). Choose a far-red fluorophore if an unstained section shows stronger autofluorescence in shorter-wavelength channels, and assess background in every channel (standard IF practice). PHGDH has no transmembrane segment and has reported cytosolic and nucleoplasmic pools, so permeabilize for intracellular epitopes while titrating detergent to preserve cellular structure (UniProt O43175 topology; HPA: subcellular; standard IF practice).
How can I reduce diffuse DAB background without losing PHGDH signal?
Compare a no-primary control with the stained section to identify signal from secondary detection or endogenous tissue activity (standard IHC practice). The selected image used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a 30-minute peroxidase-linked secondary incubation at 37°C (datasheet A03355-1). Block endogenous peroxidase, wash consistently and adjust DAB development time on matched sections; these are general chromogenic IHC controls (standard IHC practice). If diffuse staining persists, titrate the primary antibody while checking whether a documented high-staining breast glandular compartment remains detectable (HPA: High in breast glandular cells; standard IHC practice).
How should I quantify heterogeneous PHGDH DAB staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since tissue staining is generally cytoplasmic but can include nuclei (HPA: tissue IHC). For each region, report the percentage of positive cells and an H-score: multiply the percentages at intensity grades 0–3 by their grades and sum them, giving 0–300 (standard IHC practice). Normalize positive-cell counts to all eligible cells of the same type, or positive area to viable tissue area in mm²; exclude necrosis and section edges (standard IHC practice). Use consistent retrieval, imaging and scoring thresholds across matched sections, and report cytoplasmic and nuclear scores separately (datasheet A03355-1 retrieval; standard IHC practice).
How can I distinguish true PHGDH staining from artefact?
A plausible pattern follows cellular anatomy: cytoplasmic staining is common, while nuclear staining occurs in several tissues (HPA: tissue IHC). Breast glandular cells are a documented high-staining reference, but the selected breast cancer image does not establish that every tumor cell should stain (HPA: High in breast glandular cells; datasheet A03355-1). Question signal restricted to section edges, folds, necrotic regions or luminal debris, and check no-primary and endogenous-peroxidase controls before assigning it to PHGDH (standard IHC practice). Isolated sharply membranous DAB needs corroboration because PHGDH has no annotated transmembrane segment, although HPA cell imaging reports a plasma-membrane pool (UniProt O43175 topology; HPA: subcellular).
Boster reagents
Best PHGDH / D-3-phosphoglycerate dehydrogenase IHC Antibodies
The catalog antibody has IHC images from paraffin sections of human breast cancer, mouse brain and rat brain (catalog IHC captions), plus IF data from U2OS cells (catalog IF caption).
Real IHC data
Anti-PHGDH Antibody ®
Cat # A03355-1
A03355-1 has IHC images from paraffin sections of human breast cancer, mouse brain and rat brain (catalog IHC captions). A03355-1 is listed for IF/ICC, with an IF image from U2OS cells (catalog applications; catalog IF caption).
Which to pick: Choose A03355-1 for paraffin-section IHC: its captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody in human breast cancer, mouse brain and rat brain sections (A03355-1 IHC captions); the fixative is unreported (A03355-1 IHC captions). The same SKU is listed for IF/ICC and has a U2OS IF image using 5 μg/ml primary antibody (catalog applications; A03355-1 IF caption). For work across human, mouse and rat, A03355-1 lists reactivity with all three species; its clonality is unreported (catalog reactivity; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43175 (SERA_HUMAN, D-3-phosphoglycerate dehydrogenase).
- Human Protein Atlas. PHGDH tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. PHGDH subcellular location (ICC-IF): Mainly localized to the nucleoplasm, plasma membrane and cytosol. In addition localized to the mitotic spindle and primary cilium..
- Human Protein Atlas. PHGDH antibody validation summary (4 antibodies).
- Identification of Novel Diagnostic Markers for Malignant Pleural Mesothelioma Using a Reverse Translational Approach Based on a Rare Synchronous Tumor. Diagnostics (Basel, Switzerland) 2022 — PMC8871196.
- High expression of phosphoglycerate dehydrogenase predicts poor outcome in patients with high-grade serous ovarian cancer. The oncologist 2024 — PMC11379630.
- Increased Expression of PHGDH and Prognostic Significance in Colorectal Cancer. Translational oncology 2016 — PMC4907894.
- 3-Phosphoglycerate dehydrogenase expression is regulated by HOXA10 in murine endometrium and human endometrial cells. Reproduction (Cambridge, England) 2010 — PMC3107855.
- PubMed PMID:10713460 — UniProt-cited evidence.
- PubMed PMID:11055895 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PHGDH (D-3-phosphoglycerate dehydrogenase). Expected localisation General cytoplasmic staining; nuclear in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03355-1). Validated IHC antibody: A03355-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (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 breast cancer tissue; fixative not specified (datasheet A03355-1); 2. Fixation: Image fixative and duration unreported (datasheet A03355-1); 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 A03355-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03355-1); 8. Primary antibody: Rabbit anti-PHGDH, 2-5 μg/ml (datasheet A03355-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03355-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03355-1); Caveat: Antibody staining and RNA show only medium consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; the mature chain spans residues 2–533 (UniProt); Regulation: Tissue-level regulation is not annotated (UniProt). Evidence: UniProt O43175, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm, plasma membrane and cytosol. In…, Datasheet A03355-1, PMC8871196, PMC4907894. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PHGDH/immunohistochemistry-ihc-guide.