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PRODH / Proline dehydrogenase 1, mitochondrial · IHC design guide
Design Immunohistochemistry for PRODH
Plan PRODH staining in paraffin sections with the catalog antibody’s 2–5 μg/ml IHC range (datasheet A03231-3). Compare the observed cytoplasmic pattern with tissue evidence, allowing for low consistency between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PRODH 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 staining in glandular and epithelial cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03231-3) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Spleen |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining may detect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended PRODH IHC & IF Protocols
The catalog antibody protocol is followed by 3 published PRODH IHC protocols from lung cancer, breast cancer xenograft, and glioma studies (PMC10815008; PMC9261841; PMC10814259).
| Sample | Paraffin-embedded human lung adenocarcinoma tissue; fixative not specified (datasheet A03231-3) |
| Fixation | Image fixative and duration unreported (datasheet A03231-3); 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 A03231-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03231-3) |
| Primary antibody | Rabbit anti-PRODH, 2-5 μg/ml (datasheet A03231-3) |
| Primary incubation | Overnight at 4 °C (datasheet A03231-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03231-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PRODH-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteFor the catalog antibody, start with heat-mediated EDTA retrieval at pH 8.0 (datasheet A03231-3). Each published protocol used its own antibody and retrieval conditions.
Section 2
What Is the Expected PRODH Staining Pattern?
PRODH is a mitochondrial matrix protein with no annotated transmembrane segment (UniProt O43272: location and topology). In paraffin IHC, expect a cytoplasmic pattern in epithelial and glandular cells, with high staining reported in several tissues (HPA tissue IHC: general cytoplasmic expression; High in listed cells). Interpret intensity cautiously: HPA rates the tissue pattern Approved but reports low agreement with RNA and cautions that antibodies may target proteins from multiple genes (HPA tissue IHC: reliability).
| Granular cytoplasmic staining in bronchial respiratory epithelium or colonic glandular cells. | This fits the expected compartment and cells: PRODH is in the mitochondrial matrix (UniProt O43272: location), and HPA reports High staining in both cell types (HPA tissue IHC). Chromogenic granules alone cannot prove mitochondrial identity (general IHC practice). |
| Predominantly nuclear staining, with little cytoplasmic staining, in an expected positive cell population. | Treat this as discordant with the principal location (UniProt O43272: mitochondrial matrix; HPA subcellular: mitochondria approved). HPA calls nucleoplasmic localization uncertain in ICC-IF, so nuclear IHC alone should not establish it (HPA subcellular: nucleoplasm uncertain). |
| Strong signal in spleen red pulp cells while expected positive epithelial cells stain weakly. | HPA reports PRODH staining as Not detected in red pulp cells and High in several glandular and epithelial populations (HPA tissue IHC). Check specificity and endogenous chromogen activity; an unexpected positive is not proof of PRODH expression (general IHC practice). |
| Similar faint color across tissue, empty spaces, and unrelated cell populations. | A uniform haze is less persuasive than cell-associated cytoplasmic staining (HPA tissue IHC: general cytoplasmic expression; general IHC practice). Background may arise from detection reagents, insufficient blocking, or incomplete washing; compare with an appropriate control slide (general IHC practice). |
| No signal in bronchial respiratory epithelium or colonic glandular cells. | These are reported High by HPA, so a blank result warrants a run check (HPA tissue IHC). Assess tissue preservation, retrieval conditions, antibody and detection performance using the catalog antibody's IHC-P instructions and run controls (general IHC practice). |
💡Expected PRODH appearanceCall positive when a defined epithelial or glandular population shows clear cytoplasmic, potentially granular staining with limited background (HPA tissue IHC: general cytoplasmic expression and High in listed cells; UniProt O43272: mitochondrial matrix); isolated nuclear signal or uniform haze is less convincing (HPA subcellular: nucleoplasm uncertain; general IHC practice).
| Compartment and optical resolution | The mitochondrial matrix assignment predicts intracellular cytoplasmic signal (UniProt O43272: location). HPA describes tissue IHC broadly as cytoplasmic and ICC-IF as mainly mitochondrial (HPA tissue IHC; HPA subcellular). Chromogenic IHC morphology can support this pattern but does not resolve the matrix itself (general IHC practice). |
| Tissue and cell selection | Adrenal, appendix, cervical, colonic, duodenal, and gallbladder glandular cells; bronchial respiratory epithelium; and esophageal squamous epithelium are reported High (HPA tissue IHC). Spleen red pulp cells are reported Not detected (HPA tissue IHC). Choose the named cell population when comparing controls, since whole-section color can hide where the signal occurs (general IHC practice). |
| Interpretation confidence | HPA labels tissue staining Approved while noting low consistency with RNA and possible targeting of proteins from multiple genes (HPA tissue IHC: reliability). Its listed antibody, HPA020361, has IHC Approved status, whereas the antibody record does not claim IHC Enhanced validation (HPA antibodies). Treat a plausible pattern as supporting evidence, especially when staining conflicts with the listed cells (HPA tissue IHC). |
| Isoforms and antibody coverage | UniProt lists three PRODH isoforms, but the supplied record does not map the catalog antibody's epitope to them (UniProt O43272: isoforms; supplied antibody record). Do not infer which isoform a positive cell contains or explain a negative cell by isoform loss without epitope information (general IHC interpretation). |
| IF/ICC Q: Which location can corroborate IHC? | A: HPA rates mitochondrial ICC-IF localization approved; nucleoplasm and vesicles are uncertain, with a multiple-gene targeting caution (HPA subcellular). Use that finding to assess compartment plausibility, while interpreting the paraffin chromogenic result on its own IHC evidence (HPA tissue IHC: general cytoplasmic expression). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank throughout the run. | A failed stain is possible when cells reported High show no color (HPA tissue IHC: bronchial respiratory epithelium and colonic glandular cells). | Check that a run control developed; review the catalog antibody's IHC-P instructions for retrieval, dilution, and detection, then repeat with the named positive tissue (general IHC practice). Do not assign PRODH absence from a failed control. |
| The positive tissue stains, but the pattern is mostly nuclear. | The predominant location conflicts with mitochondrial matrix PRODH (UniProt O43272: location); HPA's nucleoplasmic ICC-IF signal is uncertain (HPA subcellular). | Review staining and counterstain controls, inspect cytoplasmic signal in the same cells, and seek an independent specificity check before scoring nuclear staining as PRODH (general IHC practice; HPA tissue IHC: multiple-gene caution). |
| Spleen red pulp cells show strong chromogenic signal. | That population is reported Not detected (HPA tissue IHC). Endogenous enzyme activity or nonspecific reagent staining can resemble a positive reaction (general IHC practice). | Compare an appropriate detection control, confirm the endogenous activity block for the chosen chromogen system, and review whether the color is confined to cells (general IHC practice). |
| Color spreads across cells and surrounding tissue. | Diffuse color lacks the defined cytoplasmic pattern reported by HPA (HPA tissue IHC: general cytoplasmic expression); excess reagent or inadequate washing can raise background (general IHC practice). | Compare the control slide, verify blocking and wash steps, and adjust antibody concentration only within the catalog IHC-P instructions (general IHC practice). Reassess named positive cell populations after background falls (HPA tissue IHC). |
| Brain or skeletal muscle RNA enrichment is used to predict the strongest IHC control. | HPA lists RNA as tissue enhanced in brain, choroid plexus, and skeletal muscle, but reports low RNA–staining consistency (HPA tissue IHC: RNA specificity and reliability). | Base the initial IHC control on an HPA High staining cell population, such as bronchial respiratory epithelium, and score its cells directly (HPA tissue IHC; general IHC practice). |
| Two plausible tissues give different staining strengths. | HPA reports High staining in specific epithelial and glandular populations, while UniProt describes a broader tissue expression distribution (HPA tissue IHC; UniProt O43272: tissue specificity). | Compare equivalent cell types and run controls before attributing the difference to PRODH abundance (general IHC practice). Record the compartment and background separately from intensity; HPA cautions about low RNA–staining agreement and multiple-gene targeting (HPA tissue IHC: reliability). |
Experiment evidence
IHC & IF images showing PRODH staining
A03231-3 · IHCIHC analysis of PRODH using anti-PRODH antibody (A03231-3). PRODH was detected in a paraffin-embedded section of human lung adenocarcinoma t
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100033-PRODH/tissue/cerebral+cortex#imid_12817311
HPA · HippocampusHuman Protein Atlas IHC, Hippocampus. Source: https://www.proteinatlas.org/ENSG00000100033-PRODH/tissue/hippocampal+formation#imid_12817304
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100033-PRODH/tissue/colon#imid_12817245
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100033-PRODH/tissue/liver#imid_12817238
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA020361; green = PRODH, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA020361; green = PRODH, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PRODH IHC & IF
🧪Run bronchus first and expect staining in respiratory epithelial cells (HPA: High in bronchus respiratory epithelial cells). Use spleen red pulp as the negative tissue (HPA: Not detected in spleen red pulp); cells outside the stained respiratory epithelium on the bronchus slide should be assessed for background staining, without assuming they are PRODH-negative (standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Spleen (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PRODH in A-549, HaCaT, OE19, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and PRODH-knockout tissue as a biological negative if available (hero IHC caption: rabbit primary antibody; standard IHC controls). Block endogenous peroxidase and inspect inflammatory cells for residual chromogen signal on the bronchus section (hero IHC caption: peroxidase detection with DAB; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03231-3 caption does not state the fixative (hero IHC caption: fixative not stated). The reported paraffin-section procedure uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required or whether frozen sections or IF are easier (hero IHC caption: heat retrieval in EDTA, pH 8.0). Interpret respiratory epithelial staining against background in adjacent cells and the spleen control (HPA: High in bronchus respiratory epithelial cells; HPA: Not detected in spleen red pulp; standard IHC practice).
HPA tissue IHC evidence for PRODH
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PRODH IHC Tips
Troubleshoot PRODH chromogenic IHC by checking retrieval, compartment, cell type, controls and scoring before interpreting staining intensity.
Which retrieval condition should I start with for PRODH paraffin-section IHC?
Start with heat-mediated retrieval in EDTA buffer at pH 8.0 (datasheet A03231-3). The selected paraffin-section example used that condition, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A03231-3). If staining is weak, vary heating time or try a citrate-buffer condition as a fallback, changing one variable at a time and keeping a positive control on the same run. Compare signal in respiratory epithelial cells, reported as high by HPA, with cells in splenic red pulp, reported as not detected (HPA tissue IHC).
How should I troubleshoot fixation-related loss of PRODH staining?
Target-specific fixation sensitivity is unknown: the selected image documents a paraffin-embedded lung adenocarcinoma section but does not state its fixative (datasheet A03231-3). Record the fixative, fixation duration and processing history for each specimen before comparing staining across blocks. On serial sections, keep retrieval and antibody concentration constant while comparing specimens with documented processing; include a positive control in every run. General IHC practice is to investigate prolonged fixation or inadequate fixation when morphology and staining change together, without assigning a PRODH-specific effect. Neither mitochondrial matrix localisation nor annotated acetyllysines establish a fixation response (UniProt O43272).
What staining pattern is consistent with PRODH in a paraffin section?
Expect cytoplasmic staining with a granular mitochondrial pattern when resolution and chromogen deposition permit; PRODH is assigned to the mitochondrial matrix (UniProt O43272), while HPA describes general cytoplasmic tissue staining (HPA tissue IHC). At routine bright-field resolution, do not require individual mitochondria to be visible. Score the cytoplasmic compartment separately from nuclear staining and compare serial sections with a mitochondrial marker where localisation is uncertain. HPA reports additional nucleoplasmic and vesicular localisation as uncertain, with a cross-gene antibody caution (HPA subcellular). Predominantly nuclear signal therefore needs independent validation before interpretation as PRODH.
Could isoforms or epitope accessibility explain discordant PRODH staining?
PRODH has 3 annotated isoforms, but the supplied record does not map this antibody’s epitope to any one of them (UniProt O43272; datasheet A03231-3). Check the antibody’s documented immunogen or epitope before attributing discordant staining to isoform expression. The record places PRODH in the mitochondrial matrix and annotates no transmembrane segment, so access to a matrix epitope may depend on processing and retrieval (UniProt O43272). Acetyllysines at residues 368 and 486 are annotated, but no evidence here shows that either changes antibody binding (UniProt O43272). Compare a second epitope-specific antibody, if available, on matched sections.
How can I check the IHC pattern with a separate IF experiment?
Design IF as an independent assay: the selected antibody evidence describes paraffin-section chromogenic IHC, not an IF protocol (datasheet A03231-3). Multiplex a PRODH channel with an epithelial marker in bronchial samples, where HPA reports high staining in respiratory epithelial cells, and consider a mitochondrial marker to assess distribution (HPA tissue IHC; UniProt O43272). Choose a far-red fluorophore if the specimen has substantial green-channel autofluorescence, and acquire single-stain controls to check bleed-through. Because PRODH is assigned to the mitochondrial matrix, test permeabilisation sufficient to reach a matrix epitope while preserving morphology (UniProt O43272). Include secondary-only and no-primary controls.
How do I distinguish PRODH signal from chromogenic background?
The selected example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, an anti-rabbit peroxidase secondary for 30 minutes at 37°C, and DAB detection (datasheet A03231-3). If staining is diffuse, first inspect no-primary and secondary-only controls, then titrate the primary while keeping retrieval constant. Use a peroxidase block as a general chromogenic IHC step and compare DAB development times across control and test sections. Examine pigment, folds, section edges and necrotic areas before calling signal positive. HPA warns of low staining–RNA consistency and potential cross-gene antibody recognition, so staining alone has limited specificity (HPA tissue IHC).
What is a reproducible way to quantify PRODH IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic scoring compartment before reviewing outcomes; PRODH is assigned to the mitochondrial matrix, and HPA describes general cytoplasmic tissue staining (UniProt O43272; HPA tissue IHC). For each region, record the percentage of positive cells and intensity on a 0–3 scale; an H-score sums intensity-weighted percentages and spans 0–300. If measuring positive-cell density per mm², normalise to viable tissue area and report cell-type composition rather than pooling unlike regions. Keep illumination, threshold, section thickness and DAB development consistent across slides. Exclude folds, edges and necrosis using rules set before scoring.
When should apparent PRODH positivity be treated as an artefact?
A credible result should fit a cytoplasmic mitochondrial distribution in the relevant cells: UniProt assigns PRODH to the matrix, and HPA reports high staining in bronchial respiratory epithelial cells (UniProt O43272; HPA tissue IHC). Question predominantly nuclear staining because HPA labels nucleoplasmic localisation uncertain and cautions that its antibodies may recognise proteins from multiple genes (HPA subcellular). Treat signal confined to section edges, folds or necrosis as a processing concern, and check no-primary controls for endogenous peroxidase or DAB deposition. Compare matched positive and negative control tissues within the same run. Do not infer protein abundance from a single stained field: HPA reports low consistency between staining and RNA expression (HPA tissue IHC).
Boster reagents
Best PRODH / Proline dehydrogenase 1, mitochondrial IHC Antibodies
A03231-3 has real IHC data from a paraffin-embedded human lung adenocarcinoma section (A03231-3 IHC image caption). Human, mouse, and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-PRODH Antibody ®
Cat # A03231-3
A03231-3 is the only card and lists IHC as an application, with human, mouse, and rat reactivity (catalog applications; catalog reactivity). Its image documents IHC on a paraffin-embedded human lung adenocarcinoma section (A03231-3 IHC image caption).
Which to pick: For tissue IHC, choose A03231-3: its paraffin-section image documents 2 μg/ml primary antibody after EDTA retrieval at pH 8.0 (A03231-3 IHC image caption). No SKU is listed for IF/ICC validation or has an IF image, so there is no validated IF/ICC choice here (catalog applications; IF image alts). For cross-species work, A03231-3 lists human, mouse, and rat reactivity, but its IHC image documents human tissue only; the fixative is unreported (catalog reactivity; A03231-3 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43272 (PROD_HUMAN, Proline dehydrogenase 1, mitochondrial).
- Human Protein Atlas. PRODH tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PRODH subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nucleoplasm and vesicles. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. PRODH antibody validation summary (1 antibodies).
- Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence. International journal of molecular sciences 2024 — PMC10815008.
- Tea polyphenol epigallocatechin-3-gallate inhibits cell proliferation in a patient-derived triple-negative breast cancer xenograft mouse model via inhibition of proline-dehydrogenase-induced effects. Journal of food and drug analysis 2021 — PMC9261841.
- Proline Metabolism in WHO G4 Gliomas Is Altered as Compared to Unaffected Brain Tissue. Cancers 2024 — PMC10814259.
- l-Proline Alleviates Kidney Injury Caused by AFB1 and AFM1 through Regulating Excessive Apoptosis of Kidney Cells. Toxins 2019 — PMC6521284.
- PubMed PMID:9385373 — UniProt-cited evidence.
- PubMed PMID:10192398 — UniProt-cited evidence.
- PubMed PMID:15662599 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PRODH (Proline dehydrogenase 1, mitochondrial). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); mitochondrial matrix localization (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03231-3). Validated IHC antibody: A03231-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Spleen. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung adenocarcinoma tissue; fixative not specified (datasheet A03231-3); 2. Fixation: Image fixative and duration unreported (datasheet A03231-3); 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 A03231-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03231-3); 8. Primary antibody: Rabbit anti-PRODH, 2-5 μg/ml (datasheet A03231-3); 9. Primary incubation: Overnight at 4 °C (datasheet A03231-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining may detect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Expression varies by tissue (UniProt). Evidence: UniProt O43272, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A03231-3, PMC10815008, PMC9261841. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PRODH/immunohistochemistry-ihc-guide.