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MPI / Mannose-6-phosphate isomerase · IHC design guide
Design Immunohistochemistry for MPI
Plan chromogenic MPI IHC in paraffin sections using the expected cytoplasmic pattern (HPA tissue IHC). Compare staining across cell types, including strongly stained lung macrophages (HPA tissue IHC), while keeping fixation consistent between sections (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MPI 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in varied cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M00175-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+3 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 | Lung macrophages may dominate local staining (HPA tissue IHC) | |
| Regulation | Broad tissue expression; intensity varies (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unreported (UniProt) |
Section 1
Recommended MPI IHC & IF Protocols
The catalog antibody uses heat-mediated EDTA retrieval at pH 8.0 (datasheet M00175-2). One published MPI IHC protocol stains a human prostate cancer tissue microarray (PMC9491030).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M00175-2) |
| Fixation | Image fixative and duration unreported (datasheet M00175-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M00175-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00175-2) |
| Primary antibody | Mouse monoclonal (clone 5G5) anti-MPI, 2μg/ml (datasheet M00175-2) |
| Primary incubation | Overnight at 4 °C (datasheet M00175-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M00175-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MPI-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 noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M00175-2); the published protocol does not specify retrieval conditions (PMC9491030).
Section 2
What Is the Expected MPI Staining Pattern?
MPI is a cytoplasmic protein without a transmembrane segment (UniProt P34949: location and topology). Expect cytoplasmic IHC staining in several cell types, especially adrenal glandular cells, lung macrophages, pancreatic exocrine glandular cells, salivary glandular cells and testicular Leydig cells (HPA tissue IHC: High). HPA rates its tissue IHC profile Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).
| Distinct cytoplasmic staining in glandular cells or lung macrophages. | This fits the reported general cytoplasmic profile. High staining in adrenal, pancreatic or salivary glandular cells, and in lung macrophages, offers useful positive patterns for comparison (HPA tissue IHC: profile and High). |
| Predominantly nuclear, sharply membranous or extracellular staining in tissue sections. | Treat a dominant signal outside the cytoplasm as suspect and inspect controls and morphology (UniProt P34949: Cytoplasm; HPA tissue IHC: general cytoplasmic expression). ICC-IF also reports some plasma membrane localization, so membrane signal alone does not prove an artefact (HPA subcellular: additional location). |
| Strong staining in adipocytes, parathyroid glandular cells, smooth muscle cells or soft tissue fibroblasts. | HPA reports MPI as Not detected in these cells. Review the signal for antibody cross-reactivity or endogenous chromogen-generating activity before assigning MPI positivity (HPA tissue IHC: Not detected; general IHC practice). |
| Broad, nearly uniform color over cells and surrounding section. | This gives little confidence in cell-specific MPI staining. Compare with an appropriate detection control and inspect the positive tissue pattern; nonspecific staining or endogenous detection activity can produce diffuse color (general IHC practice; HPA tissue IHC: cell-specific levels). |
| No cytoplasmic signal in a chosen HPA High cell population. | Check that the expected cells are present, then review antibody dilution, detection and antigen retrieval as general IHC workflow variables (HPA tissue IHC: High; general IHC practice). An absent stain alone does not establish absent MPI. |
💡Expected MPI appearanceCall a section positive when identifiable HPA High cell populations show convincing cytoplasmic staining; dominant nuclear or cell-free color is suspect (HPA tissue IHC: High and profile; UniProt P34949: Cytoplasm; general IHC practice).
| Tissue and cell selection | HPA reports High staining in specified glandular cells, lung macrophages and Leydig cells, but Not detected in several other cell populations. Score the annotated cell type rather than the entire tissue uniformly (HPA tissue IHC: cell levels). |
| Strength of evidence | HPA rates tissue IHC Approved with medium consistency against RNA expression. Its low tissue RNA specificity does not guarantee equal protein staining in every cell; use the observed cell-level IHC pattern for slide interpretation (HPA tissue IHC: reliability, RNA specificity and levels). |
| Intracellular location | UniProt places MPI in the cytoplasm and lists no transmembrane segment. HPA tissue IHC describes general cytoplasmic expression; a dominant nuclear or extracellular pattern calls for review (UniProt P34949: location and topology; HPA tissue IHC: profile). |
| Isoforms and processing | UniProt lists two isoforms and a chain spanning residues 2–423, but these records do not specify antibody epitope coverage or isoform-specific tissue staining. Do not assign different IHC patterns to individual isoforms from this evidence (UniProt P34949: isoforms and processing). |
| IF/ICC Q&A: where should MPI appear? | Mainly in the cytosol, with additional plasma membrane localization in HPA ICC-IF images. That approved ICC-IF finding is context for IF interpretation, not a tissue IHC scoring rule (HPA subcellular: main and additional locations). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in adrenal, pancreatic or salivary glandular cells selected as a positive comparison. | The expected cells may be absent from the section, or a general IHC step may have failed (HPA tissue IHC: High; general IHC practice). | Verify cell identity and section quality; review retrieval, primary antibody dilution and detection with an appropriate control (general IHC practice). |
| Only nuclei stain in otherwise intact tissue. | A nuclear-dominant result conflicts with the reported cytoplasmic tissue pattern (UniProt P34949: Cytoplasm; HPA tissue IHC: profile). | Check morphology and a detection control; interpret nuclear-only color cautiously rather than scoring it as MPI (general IHC practice). |
| Adipocytes or smooth muscle cells stain strongly. | Those cell populations are reported as Not detected, making the assignment uncertain (HPA tissue IHC: Not detected). | Compare the suspect signal with a control lacking primary antibody and with an HPA High cell population on a separate section (general IHC practice; HPA tissue IHC: High). |
| Color spreads across the section or obscures cell borders. | Diffuse nonspecific staining or endogenous chromogen-generating activity can obscure the reported cell-level pattern (general IHC practice; HPA tissue IHC: levels). | Review blocking, washes and detection controls; score MPI only where identifiable cells show interpretable staining (general IHC practice). |
| A medium or low population appears weaker than the positive comparison. | HPA reports Medium in breast glandular cells and bronchial respiratory epithelial cells, and Low in several other populations (HPA tissue IHC: levels). | Compare like cell types and avoid treating weaker color alone as assay failure; use an HPA High population to check the run (HPA tissue IHC: levels; general IHC practice). |
| A tissue slide shows strong membrane-edge staining with little cytoplasmic color. | HPA describes general cytoplasmic tissue staining, while its additional plasma membrane finding comes from ICC-IF (HPA tissue IHC: profile; HPA subcellular: additional location). | Review morphology and detection controls, then prioritize the tissue IHC pattern when assigning a chromogenic section result (general IHC practice; HPA tissue IHC: profile). |
Experiment evidence
IHC & IF images showing MPI staining
M00175-2 · IHCIHC analysis of MPI using anti-MPI antibody (M00175-2). MPI was detected in paraffin-embedded section of human lung cancer tissue. Heat medi
M00175-2 · IHCIHC analysis of MPI using anti-MPI antibody (M00175-2). MPI was detected in paraffin-embedded section of human breast cancer tissue. Heat me
M00175-2 · IFIF analysis of MPI using anti-MPI antibody (M00175-2). MPI was detected in immunocytochemical section of T-47D cells. Enzyme antigen retriev
A00175 · IHCIHC analysis of MPI using anti-MPI antibody (A00175). MPI was detected in paraffin-embedded section of human intestinal cancer tissues. Heat
A00175 · IHCIHC analysis of MPI using anti-MPI antibody (A00175). MPI was detected in paraffin-embedded section of human lung cancer tissues. Heat media
M00175 · IFIF analysis of MPI using anti-MPI antibody (M00175). MPI was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000178802-MPI/tissue/cerebral+cortex#imid_2312160
HPA · HippocampusHuman Protein Atlas IHC, Hippocampus. Source: https://www.proteinatlas.org/ENSG00000178802-MPI/tissue/hippocampal+formation#imid_2312161
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000178802-MPI/tissue/colon#imid_2312102
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000178802-MPI/tissue/liver#imid_2312103
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007200; green = MPI, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007200; green = MPI, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MPI IHC & IF
🧪Run lung first: macrophages should stain strongly (HPA: High in lung macrophages). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the lung slide, cells outside the macrophage population should lack comparable signal, but should not be assumed to be MPI-negative (HPA: High in lung macrophages).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MPI in A-431, HUVEC/TERT2, U-251MG, Sperm, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched mouse isotype control (caption: mouse anti-MPI primary). A verified MPI knockout sample is a biological negative control; check lung macrophages for endogenous peroxidase signal in chromogenic IHC and autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected M00175-2 tissue-IHC caption does not state a fixative (caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the paraffin-section example; whether retrieval is required has not been established (caption: heat-mediated EDTA retrieval). Frozen-section and IF workflows are not shown to be easier by the supplied evidence; interpret lung signal against macrophage-associated background (HPA: High in lung macrophages; standard IHC/IF practice).
HPA tissue IHC evidence for MPI
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MPI IHC Tips
Troubleshoot MPI staining in paraffin sections by checking retrieval, cell type, subcellular pattern and detection controls before comparing signal intensity.
How should I retrieve MPI in paraffin sections with weak cytoplasmic staining?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M00175-2). Keep retrieval time and heating method consistent across the comparison set, then check whether cytoplasmic signal improves in a reference section (standard IHC practice; HPA: general cytoplasmic expression). The selected antibody's image used 2 μg/mL primary antibody overnight at 4°C, so reproduce those conditions before attributing weak staining to retrieval (datasheet M00175-2). If staining remains weak, test a separately documented retrieval condition on adjacent sections and compare tissue preservation and background against the EDTA condition (standard IHC practice).
Could fixation explain inconsistent MPI staining between paraffin blocks?
The selected paraffin-section caption does not report a fixative, so MPI sensitivity to fixation type or duration is unknown for this antibody (datasheet M00175-2). Record each block's fixative and fixation time, and compare adjacent sections processed with the same EDTA pH 8.0 retrieval and 2 μg/mL primary concentration (datasheet M00175-2; standard IHC practice). Differences between blocks may reflect preanalytic handling, but staining alone cannot identify fixation as the cause (standard IHC practice). Include a consistently processed reference section in each run and assess morphology alongside cytoplasmic signal before adjusting retrieval or primary incubation (standard IHC practice; HPA: general cytoplasmic expression).
Where should MPI staining appear, and which lung cells should I compare?
Expect predominantly cytoplasmic staining: MPI is annotated as cytoplasmic, and HPA reports general cytoplasmic tissue expression (UniProt P34949; HPA: tissue IHC profile). HPA also places MPI mainly in the cytosol with an additional plasma-membrane location in cell imaging, so a membrane-associated component needs cellular context (HPA: subcellular localisation). In lung sections, identify macrophages separately because HPA reports high MPI staining in lung macrophages (HPA: lung macrophages). Score the selected lung-cancer section by cell population rather than treating every DAB-positive cell as a tumour cell, and use morphology plus a cell-type marker on a companion section when identity is uncertain (datasheet M00175-2; standard IHC practice).
Could isoforms or modified residues change what this antibody detects?
MPI has 2 annotated isoforms, while the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt P34949; datasheet M00175-2). Map the immunogen or epitope to both isoform sequences before interpreting staining as isoform-specific (standard antibody-validation practice; UniProt P34949: isoforms 1 and 2). The record lists acetylation at residue 2 and phosphorylation at residues 102 and 108, but supplies no evidence that these modifications alter IHC detection (UniProt P34949). MPI has no annotated transmembrane segment or glycosylation site, so neither feature establishes an extracellular epitope for this antibody (UniProt P34949).
How can I check MPI localisation by multiplex immunofluorescence?
On the separate IF/ICC guide, pair MPI with a macrophage marker such as CD68 when examining lung cells, because lung macrophages show high MPI staining in tissue (HPA: lung macrophages; standard immunophenotyping practice). Select spectrally separated dyes and consider a far-red MPI channel if tissue autofluorescence obscures shorter wavelengths; include single-stain and unstained controls (standard IF practice). Permeabilise for a cytosolic epitope, since MPI is mainly cytosolic, but establish the antibody's epitope before making claims about surface accessibility (HPA: subcellular localisation; standard IF practice). Compare diffuse cytosolic signal with any membrane-associated signal, an additional location reported by HPA (HPA: subcellular localisation).
What should I check when the DAB signal is diffuse or patchy?
The selected assay used biotinylated secondary antibody, streptavidin–biotin detection and DAB, making detection-system controls relevant to diffuse colour (datasheet M00175-2; standard IHC practice). Run a no-primary section, block endogenous peroxidase before DAB development, and assess endogenous biotin if signal persists with the biotin-based system (standard IHC practice). Compare sections blocked with 10% goat serum, as in the selected image, while keeping the 2 μg/mL primary incubation consistent (datasheet M00175-2). Examine tissue edges, folds and poorly preserved areas separately from intact cells, and compare staining with the expected cytoplasmic pattern before calling it MPI-positive (standard IHC practice; HPA: general cytoplasmic expression).
How should I score MPI staining across different cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, because HPA reports high staining in lung macrophages but medium staining in bronchial respiratory epithelial cells (HPA: tissue IHC). For tumour-cell staining, record the percentage positive and intensity, or calculate an H-score from intensity categories 0–3 for a 0–300 scale (standard IHC scoring practice). Report macrophage density separately as positive cells per mm² of evaluable tissue rather than adding those cells to a tumour-cell H-score (standard image-analysis practice; HPA: lung macrophages). Normalise comparisons to the same scored cell type, viable area, staining run and exposure or scanning settings (standard IHC practice).
How do I distinguish genuine MPI staining from misleading positive areas?
Give greatest weight to staining within intact cell cytoplasm, consistent with MPI's cytoplasmic annotation and HPA's tissue profile (UniProt P34949; HPA: general cytoplasmic expression). Identify lung macrophages before attributing their strong signal to adjacent tumour cells, since HPA reports high MPI in those macrophages (HPA: lung macrophages). Treat isolated nuclear signal, tissue-edge gradients and necrotic deposits as suspect until they reproduce in intact cells and controls (standard IHC interpretation practice; UniProt P34949: cytoplasm). Check a no-primary control and the peroxidase block when DAB appears outside plausible cells, particularly with the selected biotin-based detection method (datasheet M00175-2; standard IHC practice).
Boster reagents
Best MPI / Mannose-6-phosphate isomerase IHC Antibodies
Anti-MPI antibodies have IHC images from human paraffin-embedded cancer tissues and rat brain, and IF images from human cells (catalog image captions).
Real IHC data
Anti-MPI ® Antibody (monoclonal, 5G5)
Cat # M00175-2
Real IHC data
Anti-Mannose Phosphate Isomerase/MPI Antibody ®
Cat # A00175
Real IF data
Anti-MPI Antibody ® (monoclonal, 11I4)
Cat # M00175
M00175-2 has IHC images from human paraffin-embedded lung, breast, renal clear cell carcinoma and ovarian serous adenocarcinoma tissues, plus IF in T-47D cells (M00175-2 image captions); A00175 has IHC images from human paraffin-embedded intestinal and lung cancer tissues and rat brain (A00175 image captions). M00175 has an IF image from MCF-7 cells and lists human IF/ICC reactivity (M00175 image caption; catalog applications and reactivity).
Which to pick: Choose monoclonal M00175-2 for human paraffin-section IHC: its caption documents EDTA retrieval at pH 8.0 and 2 μg/mL primary antibody; the fixative is unreported (M00175-2 IHC image caption). Choose monoclonal M00175 for human IF/ICC, supported by its MCF-7 IF image; M00175-2 also lists human IF/ICC and has a T-47D IF image (M00175 and M00175-2 catalog applications, reactivity and IF image captions). For cross-species selection, A00175 lists human, mouse and rat reactivity and has paraffin-section IHC images for human and rat samples; its IHC captions do not report a fixative (A00175 catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P34949 (MPI_HUMAN, Mannose-6-phosphate isomerase).
- Human Protein Atlas. MPI tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MPI subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane..
- Human Protein Atlas. MPI antibody validation summary (1 antibodies).
- Mannose inhibits the growth of prostate cancer through a mitochondrial mechanism. Asian journal of andrology 2022 — PMC9491030.
- Mannose Impairs Lung Adenocarcinoma Growth and Enhances the Sensitivity of A549 Cells to Carboplatin. Cancer management and research 2020 — PMC7648561.
- Detection of PrP(BSE) and prion infectivity in the ileal Peyer's patch of young calves as early as 2 months after oral challenge with classical bovine spongiform encephalopathy. Veterinary research 2017 — PMC5738053.
- Ex vivo magnetic particle imaging of vascular inflammation in abdominal aortic aneurysm in a murine model. Scientific reports 2020 — PMC7381631.
- PubMed PMID:8307007 — UniProt-cited evidence.
- PubMed PMID:10980531 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MPI (Mannose-6-phosphate isomerase). Expected localisation General cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M00175-2). Validated IHC antibody: M00175-2 at 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M00175-2); 2. Fixation: Image fixative and duration unreported (datasheet M00175-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet M00175-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00175-2); 8. Primary antibody: Mouse monoclonal (clone 5G5) anti-MPI, 2μg/ml (datasheet M00175-2); 9. Primary incubation: Overnight at 4 °C (datasheet M00175-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Lung macrophages may dominate local staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unreported (UniProt); Regulation: Broad tissue expression; intensity varies (UniProt). Evidence: UniProt P34949, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet M00175-2, PMC9491030. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MPI/immunohistochemistry-ihc-guide.