PEX3 / Peroxisomal biogenesis factor 3 · IHC design guide

Design Immunohistochemistry for PEX3

Plan PEX3 chromogenic IHC in paraffin sections using the IHC-validated antibody (datasheet A04166). Compare granular cytoplasmic staining in glandular and respiratory epithelial cells with the tissue reference, bearing in mind its medium consistency with 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 PEX3 (IHC for PEX3): expected localisation Granular cytoplasm (HPA tissue IHC); peroxisome membrane (UniProt), antibody A04166, validated IHC image, and IHC protocol steps — Printable **PEX3** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); peroxisome membrane (UniProt), antibody A04166, controls and protocol steps. Open the full PEX3 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

PEX3 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	Granular cytoplasm (HPA tissue IHC); peroxisome membrane (UniProt)
	Staining pattern	Granular cytoplasm in glandular and respiratory epithelial cells (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A04166)
	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 tissue fixation consistent across samples. (standard IHC practice; not target-specific)
	Caveat	Staining has medium consistency with RNA expression (HPA tissue IHC)
	Regulation	No specific expression regulator reported (UniProt)
	Isoform / epitope	No annotated isoforms or processing; epitope side may matter (UniProt)

Section 1

Recommended PEX3 IHC & IF Protocols

The catalog antibody protocol is paired with one published PEX3 chromogenic IHC protocol using mouse heart sections (PMC11217276).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human uterus tissue; fixative not specified (datasheet A04166)
Fixation	Image fixative and duration unreported (datasheet A04166); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Tris-EDTA pH 9.0 (datasheet A04166); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-PEX3, 1:50 recommended; image 1:200 (datasheet A04166)
Primary incubation	Overnight at 4 °C (standard)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	PEX3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control.

💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval for the catalog antibody (datasheet A04166). The published IHC excerpt does not specify retrieval (PMC11217276).

Section 2

What Is the Expected PEX3 Staining Pattern?

PEX3 is a peroxisome membrane protein with two transmembrane segments and a long cytoplasmic region (UniProt P56589 topology). In paraffin section IHC, expect granular cytoplasmic staining in several epithelial and glandular cell populations (HPA: tissue IHC profile). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA expression (HPA: tissue IHC reliability).

What am I looking at on my slide?
Granular cytoplasmic staining in kidney tubules, colon glands, or bronchial respiratory epithelium.	This fits the reported tissue pattern and cell distribution; each listed population stains High in HPA tissue IHC (HPA: kidney, colon, bronchus). The granules are compatible with peroxisomal localization (UniProt P56589: peroxisome membrane). Chromogenic granules alone do not identify individual peroxisomes, so judge them alongside cell type and controls (general IHC interpretation).
Predominantly nuclear staining, with little granular cytoplasmic signal.	This does not match the general granular cytoplasmic tissue IHC profile (HPA: tissue IHC profile). HPA ICC-IF reports nucleoplasm as uncertain and peroxisomes as supported (HPA: subcellular localization). Do not treat nuclear staining alone as a convincing IHC positive; review background and detection controls before attributing it to PEX3 (general IHC interpretation).
Strong color in a cell population HPA reports as Not detected, especially without the expected granules.	Adipocytes, bone marrow hematopoietic cells, cervical glandular cells, and soft tissue fibroblasts are listed as Not detected (HPA: tissue IHC). Unexpected staining can reflect nonspecific binding or endogenous detection activity (general IHC practice). HPA's Supported rating and medium staining–RNA consistency do not make every cell level absolute (HPA: tissue IHC reliability).
Diffuse color across multiple compartments, extracellular areas, or most cells.	A broad haze is difficult to reconcile with the reported granular cytoplasmic profile (HPA: tissue IHC profile). Consider nonspecific primary binding, incomplete blocking or washing, and detection background (general IHC practice). Compare the slide with a control omitting primary antibody before scoring any faint granules as positive (general IHC practice).
No convincing staining in an otherwise interpretable kidney tubule or colon gland section.	Those cell populations are reported High, making them useful positive comparators for this assay (HPA: kidney and colon tissue IHC). A blank result may indicate an assay or specimen problem, but does not establish that PEX3 is absent (general IHC interpretation). Check a known positive control and the staining workflow before making a biological call (general IHC practice).

💡Expected PEX3 appearanceCall a positive when epithelial or glandular cells show clear granular cytoplasmic signal, potentially strong in HPA High populations; isolated nuclear color or diffuse haze is unconvincing (HPA: tissue IHC profile and listed cell levels; HPA: subcellular localization).

How each factor affects the staining
Cell population and tissue	HPA reports High staining in several glandular or epithelial populations, Low staining in selected neuronal and squamous populations, and Not detected in specified adipocytes, hematopoietic cells, cervical glands, and fibroblasts (HPA: tissue IHC). Choose the comparison at the cell level; a tissue name alone does not assign every cell the same expected intensity (general IHC interpretation).
Topology and compartment	PEX3 has membrane segments at residues 16–36 and 117–140, with residues 141–373 on the cytoplasmic side (UniProt P56589 topology). This supports interpreting a granular cytoplasmic pattern with peroxisomal membrane localization; it does not identify the epitope recognized by a particular antibody (UniProt P56589 topology; HPA: tissue IHC profile).
Antibody evidence	Both listed rabbit polyclonal antibodies have Supported IHC status; one also has Supported ICC status (HPA: HPA042830 and HPA058006 antibody validation). Treat that as support for reported patterns, while keeping the tissue profile's medium staining–RNA consistency in view (HPA: tissue IHC reliability). No catalog antibody dilution or retrieval condition is supplied here.
IF/ICC Q&A	Q: What localization should an IF/ICC image support? A: Peroxisomes are supported, while nucleoplasm is uncertain (HPA: subcellular localization). HPA lists ICC-IF images for A-431, U-251MG, and U2OS and Supported ICC status for HPA042830 (HPA: subcellular localization; antibody validation). Interpret IF/ICC on its dedicated guide page.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known positive tissue appears blank.	An assay or specimen issue is possible; kidney tubules and colon glands are reported High (HPA: kidney and colon tissue IHC).	Confirm the positive control, primary antibody step, detection reagents, and counterstain; then review the recorded retrieval and dilution conditions (general IHC practice). No PEX3-specific fixation sensitivity or retrieval setting is established by the supplied sources.
Only nuclei stain strongly.	The result diverges from granular cytoplasmic tissue IHC; nucleoplasmic ICC-IF localization is uncertain (HPA: tissue IHC profile; subcellular localization).	Compare with a no-primary control and assess whether cytoplasmic granules are independently visible (general IHC practice). Record nuclear-only staining as unresolved rather than calling it a confirmed PEX3 tissue pattern.
Not-detected reference cells stain strongly.	Nonspecific binding or endogenous detection activity may contribute (general IHC practice); compare the exact cell population against HPA's Not detected entries (HPA: tissue IHC).	Review a no-primary control, blocking, and detection controls; score the unexpected cells separately from nearby expected-positive cells (general IHC practice).
The section shows uniform haze that obscures granules.	Background from primary binding, blocking, washing, or detection can obscure the reported granular cytoplasmic pattern (general IHC practice; HPA: tissue IHC profile).	Compare with a no-primary control, then review blocking, wash steps, primary concentration, and chromogen development using the assay's recorded conditions (general IHC practice).
Signal is weak in a selected Low population.	Low staining is reported for hippocampal and caudate neurons, esophageal and vaginal squamous cells, ovarian follicle cells, and skin keratinocytes (HPA: tissue IHC).	Check a High reference population on a suitable control before treating weak staining as assay failure; score the Low population with the same background threshold (general IHC practice; HPA: tissue IHC cell levels).

Source: UniProt P56589 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing PEX3 staining

Immunohistochemical analysis of paraffin-embedded human uterus. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used for antigen retrieval. 3,Secondary antibody was diluted at 1:200 (room temperature, 45min).

A04166 · IHCImmunohistochemical analysis of paraffin-embedded human uterus. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000034693-PEX3/tissue/cerebral+cortex#imid_16657087

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000034693-PEX3/tissue/colon#imid_16657005

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000034693-PEX3/tissue/liver#imid_16656998

HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000034693-PEX3/tissue/pancreas#imid_16656957

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042830; green = PEX3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for PEX3 IHC & IF

🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use cervix glandular cells as the tissue negative (HPA: Not detected in cervix glandular cells); on the colon slide, compare stained glands with cells lacking punctate signal, without assuming a specific neighboring cell type is PEX3-negative.

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 PEX3 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (uncertain), Peroxisomes (supported) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus a PEX3 knockout or validated peptide-block control (standard IHC practice). For chromogenic colon IHC, block endogenous peroxidase and check endogenous biotin if using avidin–biotin detection (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative is unreported in the selected A04166 paraffin-section caption (datasheet: human uterus IHC caption). That caption uses Tris-EDTA at pH 9.0 for retrieval and 1:200 primary antibody; retrieval dependence in colon is unreported (datasheet: human uterus IHC caption). Whether frozen sections or IF are easier is unreported; assess punctate peroxisomal staining while treating nuclear signal cautiously (HPA: Peroxisomes supported; nucleoplasm uncertain), and check colon background from endogenous peroxidase (standard IHC practice).

HPA tissue IHC evidence for PEX3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →
Nasopharynx	Respiratory epithelial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Cervix	Glandular cells	Not detected	Protein (IHC)	HPA →
Soft tissue	Fibroblasts	Not detected	Protein (IHC)	HPA →

Section 3

Advanced PEX3 IHC Tips

Troubleshoot PEX3 staining in paraffin section IHC by checking retrieval, staining pattern, controls and scoring against the supplied evidence.

Which retrieval conditions should I try when PEX3 staining is weak?

Start with heat-mediated retrieval in Tris-EDTA at pH 9.0 (datasheet A04166). The selected paraffin-section example used that buffer, followed by the catalog antibody at 1:200 overnight at 4°C (datasheet A04166). If staining remains weak, vary heating duration and cooling consistently across paired sections, then assess tissue preservation and granular cytoplasmic signal together (standard IHC practice; HPA: general granular cytoplasmic pattern). A different retrieval buffer is a fallback to test on adjacent sections, with the documented pH 9.0 condition retained as the comparator (standard IHC practice; datasheet A04166). Include a known staining reference on each run so retrieval changes are distinguishable from detection failure (HPA: high in kidney tubule cells; standard IHC practice).

Could fixation explain inconsistent PEX3 staining across paraffin sections?

Target-specific PEX3 sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet A04166). Record each specimen’s fixation method and duration, section thickness and storage history before comparing staining across blocks (standard IHC practice). Process paired sections together and hold retrieval, antibody incubation and chromogenic development constant while examining whether variation follows specimen preparation (standard IHC practice; datasheet A04166: Tris-EDTA pH 9.0, antibody 1:200 overnight at 4°C). Check morphology and a same-run reference section before calling an unstained specimen biologically negative (standard IHC practice; HPA: high in kidney tubule cells).

What PEX3 pattern should I expect in chromogenic tissue sections?

Look for granular cytoplasmic staining consistent with PEX3 at the peroxisome membrane, allowing for limited resolution of individual organelles in chromogenic sections (HPA: general granular cytoplasmic pattern; UniProt P56589: peroxisome membrane; standard IHC practice). Compare the signal within the expected cell population rather than treating diffuse staining across every compartment as equivalent (standard IHC practice). High staining is reported in kidney tubule cells and bronchial respiratory epithelial cells, which can serve as pattern references when those tissues are available (HPA: high in both). Predominant nuclear staining warrants caution because the HPA nucleoplasm assignment is uncertain, whereas its peroxisome assignment is supported (HPA subcellular: nucleoplasm uncertain, peroxisomes supported).

How does PEX3 topology affect interpretation of a negative stain?

PEX3 has 2 annotated transmembrane segments, at residues 16–36 and 117–140, with its 141–373 region facing the cytoplasm (UniProt P56589 topology). Residues 37–116 face the peroxisomal side, so epitope accessibility can depend on antibody specificity and section processing (UniProt P56589 topology; standard IHC practice). The supplied evidence does not map the catalog antibody’s epitope, so a negative section cannot establish which region failed to stain (datasheet A04166: epitope not stated). No isoforms or glycosylation sites are annotated, but those annotations do not establish fixation tolerance or universal epitope preservation (UniProt P56589: isoforms 0, glycosylation sites 0; standard IHC practice).

How can I use IF to investigate an ambiguous PEX3 IHC pattern?

Use IF/ICC as a separate localisation check, pairing PEX3 with a validated marker for the cell population under examination and a peroxisome marker when testing punctate overlap (HPA: peroxisomes supported; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, and use single-label controls to assess channel bleed-through before interpreting colocalisation (standard IF practice). Match permeabilisation to the mapped antibody epitope: PEX3 residues 141–373 face the cytoplasm, whereas 37–116 face the peroxisomal side (UniProt P56589 topology). Because the catalog antibody’s epitope is unspecified, test permeabilisation conditions empirically and do not transfer the paraffin IHC retrieval conditions to IF/ICC (datasheet A04166: epitope not stated; standard IF practice).

How should I investigate diffuse or widespread PEX3 chromogenic staining?

First compare the signal with the reported granular cytoplasmic pattern and with a no-primary control processed through the same detection and development steps (HPA: granular cytoplasmic pattern; standard IHC practice). Check whether excess chromogen follows tissue edges, folds or damaged regions, then compare intact areas on adjacent sections (standard IHC practice). For peroxidase-based detection, verify the peroxidase block and assess endogenous enzyme signal with the no-primary control; these are general chromogenic workflow checks (standard IHC practice). Titrate the catalog antibody around the documented 1:200 condition while keeping retrieval at Tris-EDTA pH 9.0 and development time matched across slides (datasheet A04166; standard IHC practice).

How should I score PEX3 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and intact tissue area before scoring, then apply the same chromogenic threshold to every section (standard IHC practice). Record the percentage of positive cells and an H-score using intensity categories 0–3, or measure granular positive profiles per mm² when the image resolution supports that approach (standard IHC practice; HPA: granular cytoplasmic pattern). Normalise counts to the number of evaluable cells or tissue area in that population, and report excluded folds, edges and necrotic regions (standard IHC practice). Score comparable cell types separately because HPA reports high staining in kidney tubule cells but undetected staining in adipocytes (HPA: kidney tubule cells high; adipocytes not detected).

What makes a PEX3 positive stain convincing rather than artefactual?

A convincing stain is granular and cytoplasmic in intact cells, consistent with the reported tissue pattern and peroxisome membrane location (HPA: granular cytoplasmic pattern; UniProt P56589: peroxisome membrane). Compare it with an appropriate same-run reference: kidney tubule cells are reported high, whereas adipocytes are reported undetected by tissue IHC (HPA: kidney tubule cells high; adipocytes not detected). Treat predominant nuclear staining cautiously because the HPA nucleoplasm assignment is uncertain (HPA subcellular: nucleoplasm uncertain). Signal limited to section edges or necrotic areas, or persisting in a no-primary control after peroxidase-based detection, calls for artefact checks before biological interpretation (standard IHC practice).

Boster reagents

Best PEX3 / Peroxisomal biogenesis factor 3 IHC Antibodies

A04166 has real IHC data from paraffin-embedded human uterus (A04166 image caption) and lists human, mouse, and rat reactivity (catalog reactivity). No IF data are listed (catalog IF images).

Real IHC data Immunohistochemical analysis of paraffin-embedded human uterus. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA,pH9.0 was used for antigen retrieval. 3,Secondary antibody was diluted at 1:200 (room temperature, 45min).

Anti-Peroxin 3 PEX3 Antibody

Cat # A04166

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A04166 lists IHC as an application and human, mouse, and rat reactivity (catalog applications/reactivity). Its IHC figure shows paraffin-embedded human uterus; no IF/ICC application or figure is listed (A04166 image caption; catalog applications/IF images).

Which to pick: For tissue IHC, choose A04166, a rabbit polyclonal antibody listed for IHC with human, mouse, and rat reactivity (catalog host/dilution_raw/applications/reactivity). Its own figure documents paraffin-embedded human uterus, Tris-EDTA pH 9 retrieval, and 1:200 primary antibody overnight at 4°C; the fixative is unreported (A04166 image caption). A04166 is the only listed cross-species option by reactivity, while no SKU has listed IF/ICC validation (catalog reactivity/applications/IF images).

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

References

UniProt Consortium. UniProt entry P56589 (PEX3_HUMAN, Peroxisomal biogenesis factor 3).
Human Protein Atlas. PEX3 tissue IHC expression (reliability: Supported).
Human Protein Atlas. PEX3 subcellular location (ICC-IF): Localized to the nucleoplasm and peroxisomes..
Human Protein Atlas. PEX3 antibody validation summary (2 antibodies).
Multifunctional dual-layer microneedles loaded with selenium-doped carbon quantum dots and Astilbin for ameliorating diabetic wound healing. Materials today. Bio 2025 — PMC12022665.
PEX3 promotes regenerative repair after myocardial injury in mice through facilitating plasma membrane localization of ITGB3. Communications biology 2024 — PMC11217276.
PubMed PMID:9657383 — UniProt-cited evidence.
PubMed PMID:10430017 — UniProt-cited evidence.
PubMed PMID:10679269 — UniProt-cited evidence.