GSR / Glutathione reductase, mitochondrial · IHC design guide

Design Immunohistochemistry for GSR

Plan GSR paraffin IHC with high-staining epididymal or fallopian tube glandular cells as reference tissue (HPA tissue IHC). This guide covers the 2–5 μg/mL antibody range (datasheet A01479-1) and interpretation of cytoplasmic and nuclear staining alongside UniProt's mitochondrial and cytoplasmic annotation (HPA tissue IHC; UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for GSR (IHC for GSR): expected localisation Cytoplasmic and nuclear IHC; mitochondrial annotation (HPA tissue IHC; UniProt), antibody A01479-1, validated IHC image, and IHC protocol steps — Printable **GSR** IHC protocol sheet — expected localisation Cytoplasmic and nuclear IHC; mitochondrial annotation (HPA tissue IHC; UniProt), antibody A01479-1, controls and protocol steps. Open the full GSR IHC guide →

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GSR 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 and nuclear IHC; mitochondrial annotation (HPA tissue IHC; UniProt)
	Staining pattern	Widespread cytoplasmic and nuclear staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A01479-1)
	Positive control ⓘ	Epididymis+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific)
	Caveat	Nuclear IHC conflicts with UniProt localization (HPA tissue IHC; UniProt)
	Regulation	Regulation not annotated (UniProt)
	Isoform / epitope	Five isoforms; check the epitope against the mature chain (44–522) (UniProt)

Section 1

Recommended GSR IHC & IF Protocols

The catalog antibody’s IHC-P protocol is accompanied by two published GSR staining protocols for mouse lung sections (PMC12133717; PMC2634824).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A01479-1)
Fixation	Image fixative and duration unreported (datasheet A01479-1); 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 A01479-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A01479-1)
Primary antibody	Rabbit anti-GSR, 2-5μg/ml (datasheet A01479-1)
Primary incubation	Overnight at 4 °C (datasheet A01479-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A01479-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	GSR-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic and nuclear expression. No signal in the no-primary control.

💡Decision noteStart with the catalog antibody’s heat-mediated EDTA pH 8.0 retrieval (datasheet: A01479-1); the published mouse lung protocols used citrate pH 6.0 (PMC12133717; PMC2634824).

Section 2

What Is the Expected GSR Staining Pattern?

GSR is a soluble cytoplasmic and mitochondrial protein with no transmembrane segment (UniProt P00390: location and topology). In paraffin-section IHC, expect staining in glandular cells, especially in epididymis and fallopian tube, where HPA reports high staining (HPA: tissue IHC). HPA rates its tissue staining Approved, while noting medium agreement with RNA data and a subcellular location contradicted by external data (HPA: reliability).

What am I looking at on my slide?
Glandular cells show cytoplasmic staining, strongest in epididymis or fallopian tube.	This fits HPA's high staining in those cells and UniProt's cytoplasmic and mitochondrial locations (HPA: tissue IHC; UniProt P00390: location). A granular accent can be compatible with mitochondria, but chromogenic IHC alone cannot assign individual organelles (general IHC practice).
Staining is confined to nuclei or sharply outlines cell membranes.	Recheck specificity and detection conditions: UniProt places GSR in cytoplasm and mitochondria and lists no transmembrane segment (UniProt P00390: location and topology). HPA describes cytoplasmic and nuclear tissue staining but flags a location conflict, so nuclear signal alone is inconclusive rather than proof of an artefact (HPA: tissue IHC and reliability).
Adipocytes or cardiomyocytes stain strongly while nearby expected cells do not.	HPA reports GSR as not detected in adipocytes and cardiomyocytes in its sampled tissues (HPA: tissue IHC). This reversal raises concern for off-target binding or endogenous detection activity; it does not prove either cause (general IHC practice). Check controls before treating those cells as GSR positive.
Brown colour spreads across stroma, section edges, or many unrelated cells.	A diffuse deposit that obscures cell boundaries is difficult to score as GSR (general IHC practice). Compare a section processed without primary antibody and inspect blocking, washing, and chromogen development; HPA's tissue pattern does not establish a cause for background in this specimen (HPA: tissue IHC).
No staining appears in epididymal or fallopian-tube glandular cells.	These are useful positive reference cells because HPA reports high staining in both tissues (HPA: tissue IHC). First confirm that cells are present and the detection control worked; then review antibody dilution, retrieval, and development as general IHC variables, without assuming GSR-specific fixation sensitivity (general IHC practice).

💡Expected GSR appearanceCall a convincing positive when glandular cells show clear cytoplasmic staining, potentially high in epididymis or fallopian tube (HPA: tissue IHC; UniProt P00390: location); isolated nuclear, membrane-rim, or diffuse extracellular colour warrants control-based review (HPA: reliability; general IHC practice).

How each factor affects the staining
Compartment and interpretation	UniProt lists cytoplasm and mitochondrion, with no transmembrane segment (UniProt P00390: location and topology). HPA tissue IHC also describes nuclear staining, while its ICC-IF summary supports cytosol and approves an additional ER location (HPA: tissue IHC; HPA: subcellular). Treat compartment discrepancies as findings to investigate, not as interchangeable localisation claims.
Choice of reference cells	HPA reports high glandular-cell staining in epididymis and fallopian tube, medium staining in several other glandular epithelia, and no detected staining in sampled adipocytes or cardiomyocytes (HPA: tissue IHC). Compare the named cells within each tissue; HPA's low tissue RNA specificity does not make every cell equally useful as a control (HPA: RNA specificity).
Antibody evidence	HPA lists IHC as Approved for HPA001538 and CAB008632, while HPA064806 has no IHC status in the supplied record (HPA: antibodies). The tissue profile itself is Approved with medium RNA agreement and pending external verification (HPA: reliability). An Approved designation supports use as a reference, but does not settle every disputed compartment.
Isoforms and processing	UniProt lists five isoforms, including mitochondrial and cytoplasmic forms, and a mitochondrial chain spanning residues 44–522 (UniProt P00390: isoforms and processing). These facts help explain why cellular distribution can vary; without an epitope map for the antibody, they cannot predict which isoforms the IHC stain detects.
IF/ICC Q&A	Q: Should IF/ICC show the same pattern as paraffin IHC? A: HPA's ICC-IF summary places GSR mainly in supported cytosol, with approved additional ER staining; its tissue IHC profile also describes nuclear staining (HPA: subcellular; HPA: tissue IHC). Interpret each assay against its own controls and keep IF/ICC optimisation on its separate guide page.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A high-staining reference tissue gives little or no signal.	The section may lack the relevant glandular cells, or a general IHC detection step may have failed; HPA reports high staining specifically in epididymal and fallopian-tube glandular cells (HPA: tissue IHC).	Identify the expected cells on the counterstain, verify a working detection control, then optimise retrieval, antibody dilution, and chromogen time using the antibody's IHC instructions (general IHC practice). Do not infer a GSR-specific fixation effect from this result.
Most cells show uniform brown colour, including a no-primary control.	Background can arise from endogenous enzyme activity or the detection system rather than specific antibody binding (general IHC practice).	Review the no-primary control, apply the detection system's appropriate endogenous-activity block, and check wash and development steps (general IHC practice). Judge any remaining signal against HPA's cell-level tissue pattern (HPA: tissue IHC).
Nuclei are prominent but cytoplasm is faint.	HPA reports nuclear tissue staining yet flags a subcellular conflict; UniProt places GSR in cytoplasm and mitochondria (HPA: tissue IHC and reliability; UniProt P00390: location). The slide alone cannot resolve that conflict.	Record nuclear and cytoplasmic scores separately, inspect no-primary and tissue controls, and seek independent antibody agreement before assigning nuclear localisation (general IHC practice; HPA: reliability).
A membrane rim or extracellular deposit dominates.	That distribution conflicts with GSR's reported intracellular locations and lack of a transmembrane segment (UniProt P00390: location and topology); edge deposits can also reflect nonspecific IHC background (general IHC practice).	Compare intact cells away from section edges, review the no-primary control and washing, and require reproducible intracellular staining in reference cells before scoring GSR (general IHC practice; HPA: tissue IHC).
A supposedly negative cell population stains.	HPA's “not detected” calls apply to the sampled cells and assay, including adipocytes and cardiomyocytes; they are not proof that every specimen of that cell type lacks GSR (HPA: tissue IHC).	Check cell identity and matched controls, compare staining with an expected glandular-cell reference, and describe the finding as discordant until specificity is supported (HPA: tissue IHC; general IHC practice).
Two antibodies give different compartment patterns.	HPA's supplied antibody statuses are assay-specific, and its tissue reliability note flags a subcellular discrepancy (HPA: antibodies and reliability). Isoforms exist, but an epitope-based explanation cannot be assigned without antibody epitope data (UniProt P00390: isoforms).	Compare like-for-like IHC controls and cell types, report each antibody's pattern separately, and avoid combining nuclear, ER, and mitochondrial labels into one confirmed result (general IHC practice; HPA: subcellular; UniProt P00390: location).

Source: UniProt P00390 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing GSR staining

IHC analysis of Glutathione Reductase using anti-Glutathione Reductase antibody (A01479-1). Glutathione Reductase was detected in a paraffin-embedded section of human lung cancer 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 rabbit anti-Glutathione Reductase Antibody (A01479-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A01479-1 · IHCIHC analysis of Glutathione Reductase using anti-Glutathione Reductase antibody (A01479-1). Glutathione Reductase was detected in a paraffin

IF analysis of Glutathione Reductase using anti-Glutathione Reductase antibody (A01479-1). Glutathione Reductase was detected in an immunocytochemical section of A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Glutathione Reductase Antibody (A01479-1) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A01479-1 · IFIF analysis of Glutathione Reductase using anti-Glutathione Reductase antibody (A01479-1). Glutathione Reductase was detected in an immunocy

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000104687-GSR/tissue/cerebral+cortex#imid_891048

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000104687-GSR/tissue/colon#imid_890990

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000104687-GSR/tissue/liver#imid_890983

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000104687-GSR/tissue/kidney#imid_890984

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA064806; green = GSR, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for GSR IHC & IF

🧪Run epididymis first: glandular cells should stain (HPA: High in epididymis glandular cells). Use adipose tissue as the negative comparison, focusing on adipocytes (HPA: Not detected in adipocytes); on the positive slide, non-glandular cells should show little or no specific chromogen, but their GSR status is unreported.

Positive control tissue: Epididymis (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GSR in A-549, Hep-G2, U2OS, A-431, U-251MG, KOLF2.1J, with annotated localisation: Cytosol (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control; nonimmune rabbit IgG matched to the primary antibody’s isotype and clonality where applicable; and a GSR-knockout specimen processed alongside the test tissue (caption: rabbit primary antibody; standard IHC controls). Quench endogenous peroxidase and check for residual background in epididymis before interpreting DAB staining (caption: HRP/DAB detection; standard IHC practice).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A01479-1 caption does not state a fixative (selected-SKU tissue-IHC caption: fixative not stated). The paraffin-section example uses heat retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required for every specimen (selected-SKU tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier, and it reports no epididymis-specific artefact; assess any luminal or background DAB signal against the controls (HPA: High in epididymis glandular cells; selected-SKU tissue-IHC caption: DAB detection).

HPA tissue IHC evidence for GSR

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Subcellular location is contradicted by external 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
Epididymis	Glandular cells	High	Protein (IHC)	HPA →
Fallopian tube	Glandular cells	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Breast	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Hippocampus	Glial cells	Not detected	Protein (IHC)	HPA →
Smooth muscle	Smooth muscle cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced GSR IHC Tips

Troubleshoot GSR staining in paraffin sections by checking retrieval, staining controls and compartment patterns before comparing samples.

Which retrieval condition should I start with for GSR staining?

Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A01479-1). The selected paraffin-section image used this condition before overnight incubation with 2 μg/ml primary antibody at 4°C (caption A01479-1). If staining is weak, compare a limited range of heating times on matched sections while keeping the buffer and antibody concentration fixed; assess tissue damage alongside signal (standard IHC practice). Include the same positive control in each run so a change in retrieval can be distinguished from run variation (standard IHC practice). Record both staining intensity and preservation of cell boundaries before adopting a revised condition (standard IHC practice).

How should I troubleshoot fixation-related changes in GSR staining?

Target-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (caption A01479-1). Record fixative, fixation duration and processing history for each specimen, and compare matched sections when investigating weak or uneven staining (standard IHC practice). Start troubleshooting with the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration before changing multiple steps together (caption A01479-1; standard IHC practice). Evaluate preserved morphology and staining in internal control cells, since poorly preserved regions can give misleading intensity measurements (standard IHC practice). Do not infer a GSR-specific fixation effect from tissue staining patterns or protein annotations alone (HPA tissue IHC; UniProt P00390).

What compartment pattern should I expect in GSR IHC?

Assess cytoplasmic staining first: UniProt lists GSR in mitochondria and cytoplasm, while HPA subcellular imaging supports a mainly cytosolic location with additional endoplasmic reticulum staining (UniProt P00390; HPA subcellular). A diffuse cytoplasmic chromogenic pattern can therefore be plausible, but bright nuclear-only staining needs independent validation (UniProt P00390; HPA tissue IHC). HPA tissue IHC reports ubiquitous cytoplasmic and nuclear expression, yet flags disagreement with external subcellular data and pending verification (HPA tissue IHC). Review compartment patterns at the cell level against morphology and a no-primary control, using identical imaging settings across compared sections (standard IHC practice). Avoid assigning individual brown puncta to mitochondria from DAB morphology alone (standard IHC practice).

Can this stain distinguish mitochondrial and cytoplasmic GSR isoforms?

Do not assume that this stain separates isoforms: the supplied record lists 5 GSR isoforms, including mitochondrial and cytoplasmic forms, but provides no antibody epitope map (UniProt P00390; caption A01479-1). The annotated mitochondrial chain spans residues 44–522, and the protein has no transmembrane segment (UniProt P00390). Check the antibody's documented immunogen or epitope before interpreting compartment differences as isoform differences (standard IHC practice). Compare staining with a compartment marker on adjacent sections if isoform localisation is central to the experiment (standard IHC practice). Report the result as GSR immunoreactivity unless isoform specificity has been independently established (standard IHC practice).

How can IF help verify a GSR pattern seen by IHC?

Use IF as an independent localisation check, while treating the selected antibody's supplied tissue evidence as paraffin-section chromogenic IHC (caption A01479-1). Multiplex GSR with a marker for the cell population being assessed, such as a glandular-cell marker in a glandular compartment, and include single-label controls (HPA tissue IHC; standard IF practice). Choose a fluorophore channel after inspecting tissue autofluorescence, then acquire all comparison groups with matched settings (standard IF practice). Because GSR has no transmembrane segment and is reported in cytoplasm and mitochondria, optimise permeabilisation for intracellular epitope access without assuming where this antibody binds (UniProt P00390; standard IF practice). Confirm apparent overlap using compartment controls rather than colour merging alone (standard IF practice).

What should I check when GSR DAB staining is diffuse or excessive?

Run a no-primary control to identify secondary-reagent or tissue-derived signal, and inspect section edges and damaged areas separately (standard IHC practice). Block endogenous peroxidase before HRP detection; this is a general chromogenic workflow step, while the selected image specifically used an HRP detection system and DAB (caption A01479-1; standard IHC practice). The selected image also used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a 30-minute secondary incubation at 37°C (caption A01479-1). If background persists, titrate primary concentration and DAB development separately while retaining a positive control (standard IHC practice). Judge excess staining against cell boundaries and no-primary signal (standard IHC practice).

How should I score GSR expression across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and cytoplasmic scoring compartment before reviewing outcomes, since GSR is reported in cytoplasm and mitochondria (UniProt P00390; standard IHC practice). An H-score combines the percentage of cells at each intensity grade; alternatively report percentage of positive cells or positive-cell density per mm² within a defined region (standard IHC practice). Normalise counts to evaluable tissue area or the number of eligible cells, and apply one positivity threshold and imaging workflow across groups (standard IHC practice). Exclude folds, necrosis and section edges using preset rules, then report how many cells or regions contributed to each estimate (standard IHC practice). Keep nuclear and cytoplasmic scores separate if both are recorded (HPA tissue IHC; standard IHC practice).

How do I distinguish credible GSR staining from artefact?

A credible result follows cell morphology, repeats across intact regions and exceeds the no-primary control under the same DAB development conditions (standard IHC practice). Cytoplasmic staining is consistent with UniProt localisation, whereas an isolated nuclear-only pattern needs verification because HPA's nuclear tissue signal conflicts with external subcellular evidence (UniProt P00390; HPA tissue IHC). Compare stained cells with the expected population: HPA reports high glandular-cell signal in epididymis and fallopian tube, but no detected cardiomyocyte signal in heart muscle (HPA tissue IHC). Treat edge accentuation, necrotic deposits and residual endogenous peroxidase signal as possible artefacts until controls resolve them (standard IHC practice). Confirm surprising patterns with an independent antibody or localisation method (standard IHC practice).

Boster reagents

Best GSR / Glutathione reductase, mitochondrial IHC Antibodies

The IHC-validated antibody has paraffin-section data from human tissues and IF/ICC data from A549 cells (catalog image captions); listed reactivity covers human, mouse and rat (catalog applications/reactivity).

Real IHC data

Anti-Glutathione Reductase/GSR Antibody ®

Cat # A01479-1

View product →

A01479-1 has IHC-P images from human lung cancer, tonsil, rectal cancer and thyroid cancer paraffin sections (A01479-1 IHC image captions). It also has an IF/ICC image from A549 cells and listed reactivity with human, mouse and rat (A01479-1 IF image caption; catalog applications/reactivity).

Which to pick: Choose A01479-1 for paraffin-section IHC: its tissue captions report EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml (A01479-1 IHC image captions); the fixative is unreported (A01479-1 IHC image captions). Choose A01479-1 for IF/ICC because its A549 image documents that application (A01479-1 IF image caption). For cross-species work, A01479-1 lists mouse and rat reactivity, although the supplied IHC and IF images show human samples (catalog reactivity; A01479-1 image captions).

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

References

UniProt Consortium. UniProt entry P00390 (GSHR_HUMAN, Glutathione reductase, mitochondrial).
Human Protein Atlas. GSR tissue IHC expression (reliability: Approved).
Human Protein Atlas. GSR subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the endoplasmic reticulum..
Human Protein Atlas. GSR antibody validation summary (3 antibodies).
Distinct roles for the thioredoxin and glutathione antioxidant systems in Nrf2-Mediated lung tumor initiation and progression. Redox biology 2025 — PMC12133717.
A Novel Biological Activity of the STAT3 Inhibitor Stattic in Inhibiting Glutathione Reductase and Suppressing the Tumorigenicity of Human Cervical Cancer Cells via a ROS-Dependent Pathway. OncoTargets and therapy 2021 — PMC8275107.
Identification of Metabolic-Associated Genes for the Prediction of Colon and Rectal Adenocarcinoma. OncoTargets and therapy 2021 — PMC8020594.
Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free radical biology & medicine 2009 — PMC2634824.
PubMed PMID:2185014 — UniProt-cited evidence.
PubMed PMID:10708558 — UniProt-cited evidence.
PubMed PMID:20628807 — UniProt-cited evidence.