CHAC1 / Glutathione-specific gamma-glutamylcyclotransferase 1 · IHC design guide

Design Immunohistochemistry for CHAC1

Plan CHAC1 chromogenic IHC in paraffin sections using the catalog antibody’s pancreas example (datasheet A09017-2). Assess cytoplasmic staining in pancreatic endocrine cells and interpret intensity with the reported low consistency between staining and RNA expression (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for CHAC1 (IHC for CHAC1): expected localisation Cytoplasmic tissue staining (HPA tissue IHC), antibody A09017-2, validated IHC image, and IHC protocol steps — Printable **CHAC1** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC), antibody A09017-2, controls and protocol steps. Open the full CHAC1 IHC guide →

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CHAC1 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)
	Staining pattern	Cytoplasmic signal; high in pancreatic endocrine cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A09017-2)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Staining and RNA expression have low consistency (HPA tissue IHC)
	Regulation	UPR-linked; induction unreported (UniProt)
	Isoform / epitope	2 isoforms; epitope effects unresolved (UniProt)

Section 1

Recommended CHAC1 IHC & IF Protocols

The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet); three published CHAC1 IHC protocols provide gastric, kidney, and lung tissue examples (PMC6618068; PMC8034464; PMC11455913).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human pancreas tissue; fixative not specified (datasheet A09017-2)
Fixation	Image fixative and duration unreported (datasheet A09017-2); 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 A09017-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A09017-2)
Primary antibody	Rabbit anti-CHAC1, 1:50 recommended; image 2 μg/ml (datasheet A09017-2)
Primary incubation	Overnight at 4 °C (datasheet A09017-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A09017-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CHAC1-positive staining in glandular cells of appendix (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 (datasheet); the gastric study used citrate pH 6.0 as a published alternative (PMC6618068).

Section 2

What Is the Expected CHAC1 Staining Pattern?

In paraffin IHC, expect predominantly cytoplasmic CHAC1 staining in selected glandular, neuronal, Purkinje, pancreatic endocrine and renal tubular cells (HPA: tissue IHC, general cytoplasmic expression). CHAC1 is reported in cytosol and the trans-Golgi network and has no transmembrane segment (UniProt Q9BUX1: localization and topology). Interpret intensity cautiously: HPA rates its tissue IHC data Approved but reports low consistency between staining and RNA expression (HPA: tissue IHC reliability).

What am I looking at on my slide?
Cytoplasmic chromogen is high in pancreatic endocrine cells or kidney tubule cells (HPA: tissue IHC).	This fits the reported IHC pattern; assess the stained cell population as well as intensity. HPA reports general cytoplasmic expression, while UniProt places CHAC1 in cytosol and the trans-Golgi network (HPA: tissue IHC; UniProt Q9BUX1: localization).
Staining is mainly nuclear or extracellular, with little cytoplasmic signal in the expected cells (HPA: tissue IHC).	Treat the compartment mismatch as a possible artefact and check control sections and detection conditions (general IHC practice). The reported IHC pattern is cytoplasmic, and UniProt lists cytosol and trans-Golgi localization (HPA: tissue IHC; UniProt Q9BUX1: localization).
Strong signal dominates an unexpected cell population while expected cells show little signal (HPA: tissue IHC).	Consider cross-reactivity or endogenous detection activity; compare staining with a matched negative control (general IHC practice). HPA names specific high-staining cell populations and lists no confirmed negative tissue in this payload (HPA: tissue IHC).
Chromogen appears broadly across cells and tissue spaces without a clear cellular boundary (general IHC practice).	Diffuse background weakens a localization call. Review the negative control, blocking and washing, and whether chromogen development obscured cell boundaries (general IHC practice). HPA's reported cytoplasmic pattern alone cannot identify the source of background (HPA: tissue IHC).
A sampled HPA high-staining population, such as cerebellar Purkinje cells, has no detectable signal (HPA: tissue IHC).	Check that the expected cells are present and the run controls worked, then review the IHC-validated antibody and detection workflow (general IHC practice). One blank specimen does not establish CHAC1 absence; HPA reports high Purkinje-cell staining but low agreement with RNA data (HPA: tissue IHC).

💡Expected CHAC1 appearanceCall a positive result when clear cytoplasmic chromogen marks an HPA high-staining cell population, such as pancreatic endocrine or renal tubular cells; isolated nuclear deposits or diffuse tissue-wide colour are suspect (HPA: tissue IHC; general IHC practice).

How each factor affects the staining
Cell selection	Choose a population HPA actually scores High; pancreatic endocrine cells and renal tubule cells qualify, whereas skeletal myocytes are scored Low, not negative (HPA: tissue IHC).
Evidence strength	HPA rates tissue IHC Approved and reports low consistency with RNA expression. Its two listed antibodies have Approved IHC status; the payload does not establish enhanced IHC validation (HPA: reliability and antibodies).
Protein form	UniProt lists a 222-aa chain, no signal peptide or propeptide, no transmembrane segment, and two isoforms. No antibody epitope or isoform coverage is supplied, so predict neither variant-specific staining nor retrieval behaviour (UniProt Q9BUX1: processing, topology and isoforms).
IF/ICC Q&A: must paraffin IHC show mitochondria?	No mitochondrial-only IHC requirement follows from the supplied evidence. HPA reports approved mitochondrial localization in ICC-IF, while its tissue IHC summary is general cytoplasmic expression (HPA: subcellular ICC-IF and tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in an HPA high-staining cell population (HPA: tissue IHC).	The cells may be absent from the section, or an IHC run step may have failed (general IHC practice).	Confirm cell identity and section quality, then check a known-working positive control and the catalog antibody's IHC-P instructions before adjusting the run (general IHC practice).
Signal is predominantly nuclear or extracellular (HPA: tissue IHC pattern).	This differs from HPA's general cytoplasmic IHC profile and UniProt's cytosol/trans-Golgi annotation (HPA: tissue IHC; UniProt Q9BUX1: localization).	Compare with a negative control, inspect morphology, and reassess staining conditions before scoring it as CHAC1 (general IHC practice).
Unexpected cells stain more strongly than the selected HPA high-staining cells (HPA: tissue IHC).	Cross-reactivity or endogenous detection activity is possible; HPA does not provide a negative tissue here (general IHC practice; HPA: tissue IHC).	Inspect a matched negative control; if using peroxidase detection, verify endogenous peroxidase blocking, then score each cell population separately (general IHC practice).
The whole section has a low-contrast brown haze (general IHC practice).	Nonspecific background or overdevelopment can obscure cell-localized chromogen (general IHC practice).	Compare the negative control and review blocking, washes, antibody concentration and chromogen development using the catalog IHC-P protocol (general IHC practice).
Skeletal muscle stains weakly while its RNA profile suggests enrichment (HPA: tissue IHC and RNA specificity).	HPA scores myocytes Low by IHC despite listing skeletal muscle among RNA-enhanced tissues; it reports low staining–RNA consistency (HPA: tissue IHC).	Record the protein stain and RNA result separately; use an HPA High cell population for an IHC run check (HPA: tissue IHC; general IHC practice).
An ICC-IF image appears mitochondrial, but paraffin IHC looks broadly cytoplasmic (HPA: ICC-IF and tissue IHC).	The supplied HPA summaries describe different application-level patterns; UniProt also lists cytosol and trans-Golgi localization (HPA: ICC-IF and tissue IHC; UniProt Q9BUX1: localization).	Interpret paraffin IHC against the tissue IHC pattern and its controls; assess ICC-IF localization in the separate IF/ICC guide (HPA: tissue IHC and ICC-IF; general IHC practice).

Source: UniProt Q9BUX1 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CHAC1 staining

IHC analysis of CHAC1 using anti-CHAC1 antibody (A09017-2). CHAC1 was detected in a paraffin-embedded section of human pancreas 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-CHAC1 Antibody (A09017-2) 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.

A09017-2 · IHCIHC analysis of CHAC1 using anti-CHAC1 antibody (A09017-2). CHAC1 was detected in a paraffin-embedded section of human pancreas tissue. Heat

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/cerebral+cortex#imid_11166360

HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/thyroid+gland#imid_11166320

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/colon#imid_11166286

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000128965-CHAC1/tissue/liver#imid_11166287

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042708; green = CHAC1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for CHAC1 IHC & IF

🧪Run cerebellum first and look for staining in Purkinje cells (HPA: High in Purkinje cells). HPA detects CHAC1 in all 45 scored tissues, so use no-primary and isotype controls for the negative; other cells on the cerebellum slide should show counterstain without specific chromogen where they are unstained, but they are not validated CHAC1-negative cells (HPA: no negative tissue rows; standard IHC practice).

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

Negative control tissue: None in HPA: CHAC1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CHAC1 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).

Technical controls: Run a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control alongside the test slide; use CHAC1 knockout material or a validated immunizing-peptide block as a biological specificity control if available (selected-SKU caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check for neuronal lipofuscin pigment that could be mistaken for chromogen in cerebellum (standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). That caption uses heat retrieval in EDTA at pH 8.0, providing an IHC starting condition without establishing that retrieval is required; the supplied evidence does not establish that frozen sections or IF are easier (selected-SKU caption: EDTA retrieval; HPA: ICC-IF images). In cerebellum, assess pigment separately from DAB signal, particularly in neurons (standard IHC practice).

HPA tissue IHC evidence for CHAC1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Caudate	Neuronal cells	High	Protein (IHC)	HPA →
Cerebellum	Purkinje cells	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	High	Protein (IHC)	HPA →
Epididymis	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: CHAC1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced CHAC1 IHC Tips

Troubleshoot CHAC1 staining by checking retrieval, compartment, controls and cell-specific scoring before assigning biological meaning to chromogenic signal.

What retrieval conditions should I start with for CHAC1 in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet A09017-2). The documented paraffin-section example then used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (IHC caption A09017-2). If cytoplasmic staining is weak, vary heating time in small steps while keeping buffer, detection and section thickness consistent; include a matched section processed under the original condition (standard IHC practice). Score the expected cell compartment against background on each section, since excessive heating can damage morphology or raise nonspecific staining (standard IHC practice; HPA tissue IHC: general cytoplasmic expression).

How should I troubleshoot fixation-related loss of CHAC1 staining?

The CHAC1 paraffin-section caption does not state a fixative, so target-specific fixation sensitivity is unknown (IHC caption A09017-2). Record the actual fixative and fixation duration for each specimen, then compare sections processed and retrieved together using EDTA at pH 8.0 (standard IHC practice; datasheet A09017-2). When an affected specimen stains weakly, examine preservation and background alongside a section with reliable staining before changing primary-antibody concentration (standard IHC practice). Keep tissue handling, section thickness and DAB development consistent across comparisons so differences in chromogen deposition are not mistaken for a fixation effect (standard IHC practice).

Which CHAC1 staining patterns deserve closer validation?

In paraffin sections, use cytoplasmic staining as the initial pattern to assess because tissue IHC reports general cytoplasmic expression (HPA tissue IHC). The protein is annotated in cytosol and the trans-Golgi network, with no transmembrane segment, so a purely nuclear or membrane-rim pattern warrants additional controls (UniProt Q9BUX1 localisation and topology). An approved ICC/IF annotation instead places CHAC1 in mitochondria, making precise organelle assignment from DAB alone uncertain (HPA subcellular: mitochondria, approved). Compare staining with cell morphology and a matched negative control, and confirm any claimed organelle pattern with a separately validated localisation assay (standard IHC practice).

Can this antibody distinguish CHAC1 isoforms or a masked epitope?

CHAC1 has 2 annotated isoforms, but the supplied product caption does not identify the antibody epitope or establish isoform selectivity (UniProt Q9BUX1 isoforms; IHC caption A09017-2). Treat a positive DAB reaction as CHAC1-associated staining rather than proof of either isoform unless epitope mapping or an isoform-specific control is available (standard IHC interpretation). The annotated chain spans residues 1–222, with no annotated glycosylation sites or modified residues; these annotations do not establish epitope accessibility in paraffin sections (UniProt Q9BUX1 processing and modifications). If staining varies after retrieval, compare matched sections before attributing the difference to an isoform or modification (standard IHC practice).

How can I compare CHAC1 IHC with a multiplex IF experiment?

For a separate IF experiment, multiplex CHAC1 with a marker identifying the cell population of interest, such as pancreatic endocrine cells when assessing pancreas, and include single-stain controls (HPA tissue IHC: high in pancreatic endocrine cells; standard IF practice). Choose fluorophores after checking the tissue's autofluorescence and channel bleed-through, favoring a spectrally distinct, brighter channel for the weaker signal (standard IF practice). Because CHAC1 lacks a transmembrane segment and is annotated in cytosol and trans-Golgi network, use permeabilisation appropriate for an intracellular epitope, then titrate it to preserve morphology (UniProt Q9BUX1 topology and localisation; standard IF practice). Interpret organelle overlap cautiously because the approved ICC/IF mitochondrial annotation differs from the UniProt localisation record (HPA subcellular; UniProt Q9BUX1 localisation).

What should I check when CHAC1 DAB staining is diffuse or patchy?

First inspect the no-primary control, tissue edges and poorly preserved regions to separate antibody-dependent staining from background deposition (standard IHC practice). Block endogenous peroxidase before HRP detection, use an appropriate protein block, and keep wash and DAB development conditions identical across compared sections (standard IHC practice). The documented CHAC1 example used 10% goat serum, 2 μg/ml primary antibody, an HRP-linked detection system and DAB, which provides a starting reference for this antibody (IHC caption A09017-2). If background persists, titrate the primary antibody downward and judge whether expected cytoplasmic staining remains distinguishable from the no-primary section (standard IHC practice; HPA tissue IHC: general cytoplasmic expression).

How should I quantify heterogeneous CHAC1 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and analysis area before scoring; for pancreas, pancreatic endocrine cells are a documented high-staining population (HPA tissue IHC: high in pancreatic endocrine cells; standard IHC practice). Report the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories, using the same threshold and DAB development settings across sections (standard IHC practice). Normalise positive-cell counts to the number of evaluable cells in that population, or report stained-cell density per mm² of viable tissue if cell counts are impractical (standard IHC practice). Exclude folds, edges and necrotic areas consistently, and record whether scoring was blinded to specimen group (standard IHC practice).

How can I distinguish true CHAC1 staining from artefact?

A credible CHAC1 result shows reproducible staining in an appropriate cellular compartment and cell population, with little corresponding signal in the no-primary control (HPA tissue IHC: general cytoplasmic expression; standard IHC practice). Pancreatic endocrine cells and kidney tubule cells are reported as high-staining populations, but those observations do not validate every positive cell in another specimen (HPA tissue IHC). Be cautious with a purely nuclear or membrane-rim signal because CHAC1 is annotated in cytosol and trans-Golgi network without a transmembrane segment (UniProt Q9BUX1 localisation and topology). Reject staining confined to cut edges or necrotic regions, and check endogenous peroxidase when DAB appears without primary antibody (standard IHC practice).

Boster reagents

Best CHAC1 / Glutathione-specific gamma-glutamylcyclotransferase 1 IHC Antibodies

A09017-2 has real CHAC1 IHC data from a paraffin-embedded human pancreas section; catalog reactivity covers human, mouse, and rat (A09017-2 image caption; catalog reactivity).

Real IHC data

Anti-CHAC1 Antibody ®

Cat # A09017-2

View product →

A09017-2 is the only SKU shown, with IHC staining documented in a paraffin-embedded human pancreas section (A09017-2 image caption). Its listed applications include IHC, and its listed reactivity covers human, mouse, and rat (catalog applications; catalog reactivity).

Which to pick: For tissue IHC, choose A09017-2: its own image documents staining after EDTA pH 8.0 retrieval and DAB detection in a paraffin-embedded human pancreas section; the fixative is unreported (A09017-2 image caption). No IF/ICC choice is supported because A09017-2 has no listed IF application, dilution, or image (catalog applications; catalog IF fields). For cross-species studies, A09017-2 is a polyclonal antibody listed as reactive with human, mouse, and rat, though its IHC image documents human tissue only (catalog dilution_raw; catalog reactivity; A09017-2 image caption).

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

References

UniProt Consortium. UniProt entry Q9BUX1 (CHAC1_HUMAN, Glutathione-specific gamma-glutamylcyclotransferase 1).
Human Protein Atlas. CHAC1 tissue IHC expression (reliability: Approved).
Human Protein Atlas. CHAC1 subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. CHAC1 antibody validation summary (2 antibodies).
CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi. Helicobacter 2019 — PMC6618068.
Ferroptosis-related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma. Journal of cellular and molecular medicine 2021 — PMC8034464.
Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells. FEBS open bio 2018 — PMC5881537.
CHAC1 blockade suppresses progression of lung adenocarcinoma by interfering with glucose metabolism via hijacking PKM2 nuclear translocation. Cell death & disease 2024 — PMC11455913.
PubMed PMID:16572171 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:19109178 — UniProt-cited evidence.