POR / NADPH--cytochrome P450 reductase · IHC design guide

Design Immunohistochemistry for POR

Plan POR IHC in paraffin sections using cytoplasmic staining in adrenal gland glandular cells as a positive reference (HPA tissue IHC). This guide covers fixation consistency, an IHC-validated antibody at 2–5 µg/mL (datasheet M02166-2), chromogenic detection, and cell-level scoring.

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
Immunohistochemistry protocol sheet for POR (IHC for POR): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER membrane location (UniProt), antibody M02166-2, validated IHC image, and IHC protocol steps
Printable POR IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER membrane location (UniProt), antibody M02166-2, controls and protocol steps. Open the full POR IHC guide →

POR 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); ER membrane location (UniProt)
Staining pattern General cytoplasmic staining across cell types (HPA tissue IHC)
Antigen retrieval EDTA pH 8.0 HIER, heat-mediated (datasheet M02166-2)
Positive control ⓘ Adrenal gland+4 more · see all
Negative control ⓘ Heart muscle+1 more · see all
Important caveats
Reasons your staining may differ from the expected pattern.
Fixation Keep fixation consistent across sections. (standard IHC practice; not target-specific)
Caveat Antibody staining and RNA have medium consistency (HPA tissue IHC)
Regulation Expression regulation not specified (UniProt)
Isoform / epitope No isoforms annotated; check lumenal vs cytoplasmic epitope (UniProt)
Section 1

Recommended POR IHC & IF Protocols

The catalog antibody protocol uses EDTA pH 8.0 heat retrieval (datasheet: M02166-2). One published POR IHC protocol provides additional fixation, retrieval, detection, and counterstaining details (PMC9437039).

Recommended immunohistochemistry (IHC-P) protocol parameters
SampleParaffin-embedded human esophageal squamous carcinoma tissue; fixative not specified (datasheet M02166-2)
FixationImage fixative and duration unreported (datasheet M02166-2); verify before use.
Sectioning4–5 µm sections on charged slides (standard)
DeparaffinisationXylene, graded ethanol series to water (standard)
Antigen retrievalHeat retrieval: EDTA pH 8.0 (datasheet M02166-2); 20 min, 95–100 °C (standard)
Peroxidase block3% H2O2, 10 min, room temperature (standard)
Blocking10% goat serum (datasheet M02166-2)
Primary antibodyMouse monoclonal (clone 7F5) anti-POR, 2 μg/ml (datasheet M02166-2)
Primary incubationOvernight at 4 °C (datasheet M02166-2)
DetectionStreptavidin-biotin complex (SABC), DAB chromogen (datasheet M02166-2)
CounterstainHematoxylin, blue, dehydrate and mount (standard)
Expected resultPOR-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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: M02166-2); the published protocol reports a different retrieval solution (PMC9437039).
Section 2

What Is the Expected POR Staining Pattern?

POR is an endoplasmic reticulum membrane protein with a short lumenal segment and a large cytoplasmic domain (UniProt P16435 topology). In paraffin section IHC, expect a general cytoplasmic staining pattern (HPA tissue IHC). HPA reports high staining in several glandular, respiratory epithelial, neuronal, and hematopoietic cell populations; its tissue profile is rated Enhanced, with medium consistency between staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in adrenal glandular cells, with the relevant tissue structures visible.This fits the reported high glandular cell signal and general cytoplasmic profile (HPA tissue IHC). The large cytoplasmic POR domain makes that compartment plausible (UniProt P16435 topology). Judge intensity against the cells and controls on the same slide; a positive field alone does not establish antibody specificity (general IHC practice).
A predominantly nuclear or extracellular deposit, with little cytoplasmic signal.That is discordant with the tissue IHC profile and ER membrane assignment (HPA tissue IHC; UniProt P16435 subcellular location). Treat it as a possible staining or interpretation artefact and check controls. HPA separately reports nucleoplasmic signal in ICC-IF, so an isolated nuclear IHC result needs validation rather than an automatic biological assignment (HPA subcellular ICC-IF).
Strong staining in cardiomyocytes or smooth muscle cells while expected positive cells stain.HPA reports POR as not detected in those cell types (HPA tissue IHC). Consider cross-reactivity or endogenous chromogen activity, particularly if the deposit does not follow cell boundaries (general IHC practice). Compare an adjacent section without primary antibody before assigning unexpected staining to POR (general IHC practice).
A diffuse deposit across cells, stroma, and tissue edges.This lacks the cell-associated cytoplasmic pattern reported for POR (HPA tissue IHC). Uneven reagent exposure, insufficient blocking or washing, and endogenous detection activity are general IHC possibilities, not documented POR-specific effects (general IHC practice). A primary-omission control can help separate detection background from antibody-dependent staining (general IHC practice).
No signal in adrenal glandular cells or another HPA high-staining population.That conflicts with the reported high staining in the selected positive population (HPA tissue IHC). First confirm that the expected cells are present, then check the IHC-validated antibody, retrieval, detection reagents, and a concurrent positive control (general IHC practice). A failed run cannot establish absent POR expression (general IHC practice).
💡Expected POR appearanceA convincing IHC positive is cell-associated cytoplasmic staining of high intensity in an HPA high-staining population, such as adrenal glandular cells (HPA tissue IHC), whereas nuclear-only, extracellular, or smooth-muscle-dominant deposits warrant control-based investigation (HPA tissue IHC; general IHC practice).
How each factor affects the staining
Compartment and epitope orientationPOR has one membrane span at residues 22–42 and a cytoplasmic region at 43–677 (UniProt P16435 topology). The tissue IHC readout is general cytoplasmic staining (HPA tissue IHC). Epitope position for an unspecified antibody is unknown, so topology alone cannot predict its staining strength or retrieval needs.
Choice of comparison tissueHPA reports high staining in adrenal and intestinal glandular cells, respiratory epithelial cells, cortical neuronal cells, and bone marrow hematopoietic cells (HPA tissue IHC). It reports no detection in cardiomyocytes or smooth muscle cells and low staining in skeletal myocytes (HPA tissue IHC). These are useful contrasts, subject to the stated medium staining–RNA consistency (HPA tissue IHC).
Interpretation confidenceThe tissue profile carries an Enhanced reliability label but only medium consistency between antibody staining and RNA expression (HPA tissue IHC). Two listed antibodies have Enhanced IHC status, while the third has no supplied IHC status (HPA antibodies). These labels support comparison with the reported pattern; they do not validate an unexpected compartment or cell type in a new specimen.
Processing and variantsUniProt lists a 2–677 chain, no signal peptide or propeptide, zero annotated glycosylation sites, and zero isoforms (UniProt P16435 processing and isoforms). These annotations give no basis here for a second isoform pattern or a shed, extracellular POR pool. They also do not establish how paraffin processing affects antigen detection.
Antigen retrievalTarget-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.
IF/ICC Q&A: should its pattern match paraffin IHC?No direct match can be assumed: HPA tissue IHC describes general cytoplasmic expression, whereas HPA ICC-IF reports mainly vesicles, with additional nucleoplasm and cytosol localisation (HPA tissue IHC; HPA subcellular ICC-IF). Those are distinct assay observations. The ICC-IF record does not supply an IHC protocol choice or resolve nuclear-only staining in a paraffin section.
Why is my staining missing, weak or wrong?
SituationLikely causeNext action
Known-positive glandular cells are blank throughout the run.A run-level staining failure is possible when a population reported as high by HPA is absent (HPA tissue IHC; general IHC practice).Verify the cell population and concurrent positive control; review retrieval, the IHC-validated antibody, and detection steps using standard paraffin IHC controls (general IHC practice).
Only tissue edges or broad acellular regions develop chromogen.The distribution does not resemble HPA's general cytoplasmic POR profile (HPA tissue IHC); uneven reagent coverage or background is possible (general IHC practice).Compare a primary-omission section, inspect section coverage, and review blocking and washes before scoring cell-associated signal (general IHC practice).
Cardiomyocytes or smooth muscle cells appear strongly positive.Those cell types are reported as not detected by HPA; antibody cross-reactivity or endogenous detection activity may explain discordant staining (HPA tissue IHC; general IHC practice).Check a primary-omission control and an HPA high-staining cell population on the same run; investigate the detection system if signal persists without primary antibody (general IHC practice).
Staining appears confined to nuclei.Nuclear-only tissue IHC conflicts with the general cytoplasmic profile (HPA tissue IHC), although nucleoplasm is an additional ICC-IF location (HPA subcellular ICC-IF).Check the counterstain, compare a primary-omission control, and confirm cytoplasmic staining in a known-positive tissue before interpreting nuclear IHC signal (general IHC practice).
A positive tissue stains weakly while other assay controls pass.The sampled population may differ from the HPA high-staining cell type, or IHC conditions may yield weak signal (HPA tissue IHC; general IHC practice). No POR-specific fixation sensitivity is supplied.Identify the expected cells first, then compare adjacent controlled sections under documented retrieval and antibody conditions; retain the same scoring threshold across comparisons (general IHC practice).
An IF/ICC image looks vesicular but paraffin IHC looks broadly cytoplasmic.These descriptions come from different HPA assays: vesicles are the main ICC-IF location, while tissue IHC reports general cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC).Interpret each image against its assay-specific reference and controls; use the separate IF/ICC guide for that assay rather than transferring its localisation pattern into IHC scoring (HPA tissue IHC; HPA subcellular ICC-IF).

Sample controls for POR IHC & IF

🧪Run adrenal gland first; its glandular cells should stain (HPA: High in adrenal gland glandular cells). Run heart muscle as the negative tissue (HPA: Not detected in cardiomyocytes); on the adrenal slide, assess non-glandular cells for background staining without assuming they are POR-negative (HPA: adrenal gland entry specifies glandular cells only).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show POR in A-431, U-251MG, U2OS, A-549, GAMG, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched mouse isotype controls, plus a POR-knockout specimen if available or a peptide-block control if the immunizing peptide is available (caption: mouse anti-POR antibody; standard IHC practice). Block endogenous peroxidase and assess endogenous biotin background in the adrenal section because the reported IHC detection uses a biotinylated secondary, streptavidin–biotin complex, and DAB (caption: detection method; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M02166-2 paraffin-section caption does not state the fixative (caption: fixative not stated). That caption reports heat-mediated retrieval in EDTA at pH 8.0, establishing a workable condition for the pictured section without establishing a POR-wide retrieval requirement (caption: retrieval conditions). The supplied evidence does not establish that frozen sections or IF are easier; for adrenal IHC, check peroxidase and biotin background before interpreting chromogen (caption: biotin-based DAB detection; standard IHC practice).

HPA tissue IHC evidence for POR

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

TissueCell typeLevelEvidenceSource
Adrenal gland Glandular cells High Protein (IHC) HPA →
Appendix Glandular cells High Protein (IHC) HPA →
Bone marrow Hematopoietic cells High Protein (IHC) HPA →
Bronchus Respiratory epithelial cells High Protein (IHC) HPA →
Cerebral cortex Neuronal cells High Protein (IHC) HPA →

Undetected expression · recommended negative controls

TissueCell typeLevelEvidenceSource
Heart muscle Cardiomyocytes Not detected Protein (IHC) HPA →
Smooth muscle Smooth muscle cells Not detected Protein (IHC) HPA →
Section 3

Advanced POR IHC Tips

Troubleshoot POR staining in paraffin sections by checking retrieval, compartment, controls and scoring against the reported tissue patterns (datasheet M02166-2; UniProt P16435; HPA tissue IHC).

Which retrieval conditions should I try first when POR staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet M02166-2). The paraffin-section example used this retrieval before overnight incubation at 4°C with 2 μg/ml antibody, so keep those conditions consistent while evaluating retrieval (datasheet M02166-2). Compare a known positive compartment and a no-primary control across runs, and record heating and cooling conditions because retrieval intensity can change chromogenic staining (standard IHC practice). If staining remains weak, test a different retrieval buffer or heating duration on matched sections as a fallback, while checking that background has not increased (standard IHC practice).
How should I assess whether fixation is limiting POR detection?
The selected paraffin-section caption does not state a fixative, so POR-specific fixation sensitivity is unknown (datasheet M02166-2). Record the fixative, time to fixation and fixation duration for each specimen, then compare similarly processed positive-control sections before changing the antibody concentration (standard IHC practice). If processing differs between batches, run matched sections with the same EDTA pH 8.0 retrieval and 2 μg/ml primary incubation to identify a handling-related difference (datasheet M02166-2; standard IHC practice). Treat any improvement after changing fixation as an observed result for those specimens, rather than an established POR requirement (standard IHC practice).
Where should convincing POR staining appear in a paraffin section?
Expect predominantly cytoplasmic staining by chromogenic tissue IHC, with an endoplasmic-reticulum-associated pattern consistent with POR being an ER membrane protein (HPA tissue IHC: general cytoplasmic expression; UniProt P16435: ER membrane). POR has a membrane segment at residues 22–42 and a large cytoplasmic region at 43–677, so diffuse nuclear-only DAB staining needs independent confirmation (UniProt P16435 topology; standard IHC practice). Interpret fine cytoplasmic staining at the resolution of the section rather than claiming that DAB resolves individual ER membranes (standard IHC practice). HPA also reports vesicles, nucleoplasm and cytosol in subcellular imaging, which can guide follow-up localisation experiments but does not alone establish the compartment of a tissue-IHC signal (HPA subcellular).
Can epitope position explain inconsistent POR staining?
No POR isoforms are annotated in the supplied record, and the selected antibody’s epitope is not specified, so do not assign staining differences to a particular isoform or epitope (UniProt P16435 record; datasheet M02166-2). POR spans the membrane at residues 22–42, leaving residues 43–677 on the cytoplasmic side; epitope accessibility may therefore depend on where the antibody binds (UniProt P16435 topology; standard IHC practice). The record lists N-acetylglycine at position 2 and phosphoserine at 63, but neither establishes an effect on this antibody (UniProt P16435 modified residues). If staining disagrees with controls, seek documented epitope information and compare independently validated antibodies on adjacent sections (standard IHC practice).
How should I follow up an IHC pattern with multiplex POR immunofluorescence?
Treat multiplex IF as a separate validation experiment: the selected antibody’s paraffin-section image establishes a chromogenic tissue workflow, while its fixative and IF performance are unreported here (datasheet M02166-2). Pair POR with a marker for the cell type being assessed, such as an epithelial marker when investigating glandular cells reported as strongly stained, and check each channel separately (HPA tissue IHC; standard IF practice). Choose a far-red POR channel where tissue autofluorescence interferes with shorter wavelengths, and include single-stain controls for bleed-through (standard IF practice). Because POR has a large cytoplasmic region at residues 43–677, optimise permeabilisation for a cytoplasmic-side epitope only if the antibody’s epitope location supports that choice (UniProt P16435 topology; standard IF practice).
What should I check when POR DAB staining looks widespread?
The illustrated workflow uses a 10% goat-serum block, biotinylated secondary antibody, avidin–biotin detection and DAB (datasheet M02166-2). Run no-primary and secondary-only controls, then check endogenous peroxidase activity and endogenous biotin as possible sources of chromogenic signal in that workflow (standard IHC practice). If background persists, review blocking, washing and the 2 μg/ml primary concentration on matched sections before deciding that widespread color represents POR (datasheet M02166-2; standard IHC practice). Judge candidate staining within intact cell cytoplasm against control sections, since precipitate, folds and damaged tissue can create misleading DAB deposits (HPA tissue IHC: general cytoplasmic expression; standard IHC practice).
How can I score POR IHC across specimens with different cellular composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because reported POR tissue staining is generally cytoplasmic and varies by cell type (HPA tissue IHC). For glandular or epithelial regions, report the percentage of positive cells and an H-score based on intensity categories 0–3, using the same thresholds across batches (HPA tissue IHC; standard IHC practice). If counting discrete positive cells, report density per mm² of viable, annotated tissue and exclude folds, necrosis and empty space (standard IHC practice). Normalise comparisons to the relevant cell population or viable tissue area, include a matched positive control on each run, and keep acquisition and scoring rules fixed (standard IHC practice).
How do I distinguish genuine POR positivity from staining artefact?
A credible signal should fit cytoplasmic POR expression and be evaluated in the relevant cell population: HPA reports high staining in adrenal glandular cells and no detection in cardiomyocytes (HPA tissue IHC; UniProt P16435: ER membrane). Nuclear-only staining without corresponding cytoplasmic signal warrants caution, even though nucleoplasm is an additional location in subcellular imaging (HPA subcellular; standard IHC practice). Compare the suspect region with intact tissue away from edges and necrosis, then inspect no-primary controls for deposits or endogenous peroxidase signal (standard IHC practice). Confirm unexpected cell-type staining on another section or with an independently validated antibody before interpreting it as altered POR biology (standard IHC practice).
Boster reagents

Best POR / NADPH--cytochrome P450 reductase IHC Antibodies

Anti-POR IHC images cover human esophageal squamous carcinoma, mammary cancer and placenta, plus mouse and rat tissues; an ICC image shows human HeLa cells (catalog image captions).

Real IHC data IHC analysis of POR using anti-POR antibody (M02166-2). POR was detected in a paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-POR Antibody (M02166-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.
Anti-POR Antibody ® (monoclonal, 7F5)
Cat # M02166-2
Real IHC data IHC analysis of POR using anti-POR antibody (PB9736). POR was detected in paraffin-embedded section of Human Mammary Cancer Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-POR Antibody (PB9736) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.
Anti-Cytochrome P450 Reductase/POR Antibody ®
Cat # PB9736
Real IHC data Anti-POR antibody, PA1952, IHC(P) IHC(P): Human Placenta Tissue
Anti-Cytochrome P450 Reductase/POR Antibody ®
Cat # PA1952

M02166-2 has human paraffin-section IHC images from esophageal squamous carcinoma, liver cancer, lung cancer and placenta; PB9736 has human mammary cancer and mouse and rat intestine IHC images (catalog image captions). PA1952 has human placenta and rat ovary IHC images plus a human HeLa ICC image (catalog image captions).

Which to pick: For human tissue IHC, M02166-2 is a mouse monoclonal with a paraffin-section image using EDTA retrieval at pH 8.0; the caption does not report the fixative (catalog host/clone; M02166-2 IHC caption). For cross-species tissue IHC, PB9736 lists human, mouse and rat reactivity and has paraffin-section images in all three species using citrate retrieval at pH 6; its captions do not report the fixative (catalog reactivity; PB9736 IHC captions). For IF, M02166 lists IF and human, mouse and rat reactivity but has no IF image; for image-backed ICC, PA1952 has a human HeLa ICC image (catalog applications/reactivity/image alts; PA1952 ICC caption).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

  1. UniProt Consortium. UniProt entry P16435 (NCPR_HUMAN, NADPH--cytochrome P450 reductase).
  2. Human Protein Atlas. POR tissue IHC expression (reliability: Enhanced).
  3. Human Protein Atlas. POR subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the nucleoplasm and cytosol..
  4. Human Protein Atlas. POR antibody validation summary (3 antibodies).
  5. Humanized liver TK-NOG mice with functional deletion of hepatic murine cytochrome P450s as a model for studying human drug metabolism. Scientific reports 2022 — PMC9437039.
  6. Reactivation of CDX2 in Gastric Cancer as Mark for Gene Silencing Memory. Acta histochemica et cytochemica 2015 — PMC4564377.
  7. Self-Regulated ROS Nanoplatform Enables Sequential Antibacterial Therapy and Inflammation Resolution in Periodontitis. ACS omega 2026 — PMC13557269.
  8. Integrated follicular fluid multi-omics identifies steroidogenic dysregulation and a candidate SHBG-associated rescue framework in poor ovarian response. Frontiers in endocrinology 2026 — PMC13574599.
  9. PubMed PMID:1550342 — UniProt-cited evidence.
  10. PubMed PMID:12853948 — UniProt-cited evidence.
  11. PubMed PMID:15489334 — UniProt-cited evidence.