REG3G / Regenerating islet-derived protein 3-gamma · IHC design guide

Design Immunohistochemistry for REG3G

Plan REG3G IHC-P using the catalog antibody at 1:25 (datasheet M05473). Compare staining with the reported high Paneth-cell signal in duodenum and small intestine (HPA tissue IHC), while accounting for REG3G secretion (UniProt).

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

Immunohistochemistry protocol sheet for REG3G (IHC for REG3G): expected localisation Secreted; cytoplasmic location also annotated (UniProt), antibody M05473, validated IHC image, and IHC protocol steps — Printable **REG3G** IHC protocol sheet — expected localisation Secreted; cytoplasmic location also annotated (UniProt), antibody M05473, controls and protocol steps. Open the full REG3G IHC guide →

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REG3G 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	Secreted; cytoplasmic location also annotated (UniProt)
	Staining pattern	High Paneth-cell staining; compartment unreported (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet M05473)
	Positive control ⓘ	Duodenum+2 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Use consistent formaldehyde fixation for paraffin sections (datasheet M05473 IHC-P) (selected-SKU IHC image M05473)
	Caveat	Secreted protein may appear away from its source cells (HPA tissue IHC)
	Regulation	Acute-phase associated (UniProt)
	Isoform / epitope	2 isoforms; processing may remove residues 27–37 (UniProt)

Section 1

Recommended REG3G IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with three published REG3G IHC workflows (PMC7144519; PMC5636971; PMC7583020).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Formaldehyde-fixed, paraffin-embedded human pancreas tissue (datasheet M05473)
Fixation	Image formalin-fixed; duration unreported (datasheet M05473); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6 (datasheet M05473); 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	Mouse monoclonal (clone 1790CT313.66.2) anti-REG3G, 1:25 (datasheet M05473)
Primary incubation	1 hours at 37°C (datasheet M05473)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	REG3G-positive staining in paneth cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Plasma protein. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: M05473); optimize locally because these published IHC excerpts do not specify retrieval.

Section 2

What Is the Expected REG3G Staining Pattern?

For paraffin-section IHC, expect REG3G-associated staining in Paneth cells of the duodenum and small intestine, where HPA reports high staining; appendix enterocytes show medium staining (HPA tissue IHC). REG3G is secreted and also annotated in the cytoplasm, with no transmembrane segment (UniProt Q6UW15). Interpret cellular staining cautiously: HPA rates tissue IHC Uncertain because the antibodies may recognize proteins from more than one gene (HPA tissue IHC).

What am I looking at on my slide?
Distinct staining in duodenal or small-intestinal Paneth cells, stronger than adjacent cells.	This matches the reported high Paneth-cell pattern (HPA tissue IHC). Record cell identity and distribution as well as intensity; staining alone does not resolve REG3G from another protein recognized by the antibody because the IHC evidence remains Uncertain (HPA tissue IHC).
Predominantly nuclear staining or a crisp, continuous membrane outline, with little cellular signal.	That compartment pattern is unsupported: REG3G is annotated as secreted and cytoplasmic and has no transmembrane segment (UniProt Q6UW15). Check section morphology and detection controls before interpreting it as specific; an isolated membrane outline is especially weak evidence for REG3G (general IHC practice).
Strong staining in cells outside the reported positive populations, including a listed negative cell type.	Compare the exact tissue and cell type with HPA, rather than assigning a whole tissue a single status (HPA tissue IHC). Unexpected staining may reflect antibody cross-reactivity, which HPA flags, or endogenous detection activity; use appropriate antibody and detection controls to distinguish them (HPA tissue IHC; general IHC practice).
Diffuse color across stroma, lumina, and unrelated cells, without a clear Paneth-cell pattern.	REG3G is secreted, so extracellular material cannot be dismissed solely by location (UniProt Q6UW15). Broad, uniform color that persists outside the expected cellular pattern is less persuasive; compare a no-primary control and assess nonspecific binding or detection background (HPA tissue IHC; general IHC practice).
No discernible staining in well-preserved duodenal or small-intestinal Paneth cells.	This conflicts with HPA's reported high staining in those cells, but does not by itself prove biological absence (HPA tissue IHC). Check cell identification, run controls, and review the antibody and detection workflow; HPA's uncertain IHC reliability limits conclusions from either a positive or negative result (HPA tissue IHC; general IHC practice).

💡Expected REG3G appearanceA persuasive positive result is conspicuous cellular staining in duodenal or small-intestinal Paneth cells, consistent with HPA's high level; isolated nuclear, sharply membrane-only, or indiscriminate tissue-wide color is a warning pattern rather than a REG3G call (HPA tissue IHC; UniProt Q6UW15; general IHC practice).

How each factor affects the staining
Tissue and cell selection	Use Paneth cells in duodenum or small intestine as reported high IHC references; appendix enterocytes are reported medium (HPA tissue IHC). UniProt calls pancreas the predominant expression site, but that statement does not establish a pancreatic IHC pattern (UniProt Q6UW15).
Secretion and tissue location	REG3G is annotated as secreted and cytoplasmic (UniProt Q6UW15). HPA warns that RNA and protein locations can differ for a secreted protein; do not treat pancreatic RNA enrichment as a substitute for cell-level protein staining (HPA tissue IHC).
Antibody evidence	HPA047894 and HPA048334 each have Uncertain IHC status, and HPA warns of recognition of proteins from more than one gene (HPA antibodies; HPA tissue IHC). Match the staining pattern to morphology and controls before assigning REG3G specificity (general IHC practice).
Processing and epitope coverage	UniProt lists a signal peptide at residues 1–26, a propeptide at 27–37, and chains spanning 27–175 or 38–175 (UniProt Q6UW15). Antibody epitope information is absent here, so the record cannot predict which processed form an IHC stain detects.
Isoforms and retrieval	Target-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: What localisation should an IF image show?	HPA reports approved cytosolic ICC-IF localisation, while cautioning that the antibody targets proteins from multiple genes (HPA subcellular). That IF observation supports a cytosolic comparison; it does not validate the paraffin-IHC antibody or establish an IF protocol (HPA subcellular; HPA antibodies).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Paneth cells are negative in a duodenal or small-intestinal section.	Possible cell-identification, staining-workflow, or detection failure; HPA reports high staining there but rates IHC Uncertain (HPA tissue IHC; general IHC practice).	Confirm Paneth-cell morphology and section quality, then review retrieval, primary-antibody use, detection, and run controls together; avoid calling biological absence from this slide alone (general IHC practice).
A listed negative population stains strongly.	Cross-reactivity is plausible given HPA's multi-gene warning; endogenous detection activity is another general IHC possibility (HPA tissue IHC; general IHC practice).	Identify the stained cell type, compare its HPA entry, and inspect no-primary and relevant detection controls before attributing the signal to REG3G (HPA tissue IHC; general IHC practice).
Color covers most of the section without a readable cellular pattern.	Nonspecific binding or detection background may obscure interpretation; secretion alone cannot establish that every extracellular deposit is specific (UniProt Q6UW15; general IHC practice).	Compare the no-primary control, reassess blocking and washes, and score only interpretable structures against the reported Paneth-cell pattern (HPA tissue IHC; general IHC practice).
Signal appears nuclear or confined to sharply outlined cell membranes.	Those dominant compartments are unsupported by the secreted/cytoplasmic annotation and lack of a transmembrane segment (UniProt Q6UW15).	Check morphology and detection controls, then compare the same run with a reported positive tissue; treat the discordant compartment as unresolved until specificity is supported (HPA tissue IHC; general IHC practice).
Pancreatic staining differs from what its high RNA expression suggests.	UniProt describes predominant pancreatic expression, whereas HPA warns that secreted protein and RNA locations may differ (UniProt Q6UW15; HPA tissue IHC).	Document the pancreatic cell-level pattern without assigning an expected IHC intensity from RNA; compare staining with HPA's reported positive cell populations (HPA tissue IHC; general IHC practice).
A positive stain varies between antibody preparations or cannot be assigned to one processed form.	HPA IHC antibody status is Uncertain; UniProt lists two chains, while antibody epitopes are unspecified in this payload (HPA antibodies; UniProt Q6UW15).	Report the antibody identifier, cellular pattern, and controls; keep the interpretation at REG3G-associated staining unless specificity and epitope coverage are independently established (HPA tissue IHC; general IHC practice).

Source: UniProt Q6UW15 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing REG3G staining

M05473 staining REG3G in human pancreas tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 0. 5 hour at room temperature; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

M05473 · IHCM05473 staining REG3G in human pancreas tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections).

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143954-REG3G/tissue/cerebral+cortex#imid_15611507

HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000143954-REG3G/tissue/small+intestine#imid_15604128

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143954-REG3G/tissue/colon#imid_15604161

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143954-REG3G/tissue/liver#imid_15604154

HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA060705; green = REG3G, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for REG3G IHC & IF

🧪Run duodenum first: Paneth cells should stain strongly (HPA: High in duodenal Paneth cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); neighboring non-Paneth cells on the duodenal slide should show background-level staining relative to Paneth cells (HPA: High in duodenal Paneth cells).

Positive control tissue: Duodenum (Paneth cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show REG3G in SH-SY5Y, U-251MG, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus a REG3G knockout specimen or validated peptide-block control where available (standard IHC specificity controls). Check endogenous peroxidase and biotin background in duodenum when using chromogenic detection with the captioned biotinylated secondary antibody (selected-SKU IHC caption: biotinylated secondary).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact M05473 tissue-IHC caption does not establish the fixative (selected-SKU IHC caption: fixative unreported). The caption uses heat-mediated citrate retrieval at pH 6, but retrieval dependency is unreported (selected-SKU IHC caption: citrate retrieval, pH 6). Whether frozen sections or IF are easier is unreported; interpret diffuse or luminal signal cautiously because REG3G is secreted (UniProt Q6UW15: secreted).

HPA tissue IHC evidence for REG3G

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Caution, targets protein from more than one gene. Secreted protein, tissue location of RNA and protein is expected to differ. 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
Duodenum	Paneth cells	High	Protein (IHC)	HPA →
Small intestine	Paneth cells	High	Protein (IHC)	HPA →
Appendix	Enterocytes	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced REG3G IHC Tips

Troubleshoot REG3G chromogenic IHC by checking retrieval, tissue context, and secreted protein distribution before interpreting staining intensity.

Which retrieval conditions should I try first for REG3G IHC?

Start with heat-mediated citrate buffer at pH 6 for paraffin-section chromogenic IHC (datasheet M05473). The selected pancreas image used this retrieval with primary antibody at 1/25 for 1 hour at 37°C (caption M05473). If staining is weak, adjust heating time in small increments while keeping buffer and detection conditions fixed, and inspect tissue morphology after each change (standard IHC practice). Include a known positive section in every run; weak signal across both test and control sections suggests a workflow problem before it supports a biological conclusion (standard IHC practice).

How should I troubleshoot fixation-related loss of REG3G staining?

Target-specific fixation sensitivity for REG3G is unknown from the supplied evidence; do not assign a preferred fixation duration or claim that a particular fixative preserves its epitope. The selected pancreas caption describes a formaldehyde-fixed, paraffin-embedded workflow, but supplies no comparison between fixation conditions (caption M05473). When signal varies, compare sections with documented fixation histories using the same pH 6 citrate retrieval and antibody conditions (datasheet M05473; standard IHC practice). Check whether morphology and staining of a known positive control vary together, and interpret any apparent fixation effect as provisional until reproduced under matched processing conditions (standard IHC practice).

Where should convincing REG3G staining appear in a tissue section?

REG3G is annotated as secreted and cytoplasmic, with no transmembrane segment (UniProt Q6UW15 subcellular location and topology). Therefore, evaluate both cellular staining and nearby extracellular or luminal signal rather than requiring a membrane outline (UniProt Q6UW15 subcellular location; standard IHC interpretation). The selected catalog image shows staining in human pancreas, while HPA reports high signal in duodenal and small-intestinal Paneth cells and medium signal in appendix enterocytes (caption M05473; HPA tissue IHC). Compare patterns with tissue architecture and a matched control section: extracellular staining need not identify the producing cell, and HPA marks its tissue IHC reliability uncertain (UniProt Q6UW15 secretion; HPA tissue IHC reliability).

Could processing or isoforms explain an unexpected REG3G IHC pattern?

REG3G has 2 annotated isoforms and a cleaved signal peptide at residues 1–26 (UniProt Q6UW15 isoforms and processing). Annotated chains begin at residue 27 or 38, so an antibody against a removed region could behave differently from one recognizing the retained lectin domain at residues 47–172 (UniProt Q6UW15 processing and domains). The supplied product evidence does not locate the catalog antibody’s epitope; consult its mapped immunogen before attributing staining differences to cleavage or splice variation. REG3G has 0 annotated glycosylation sites, so do not invoke a documented glycan-dependent epitope effect here (UniProt Q6UW15 glycosylation).

How can I assess REG3G by multiplex immunofluorescence?

Validate REG3G IF separately from this antibody’s chromogenic paraffin-section evidence; the selected caption documents IHC, not an IF protocol (caption M05473). Pair REG3G with an independently validated marker for the expected cell type, such as Paneth cells in small intestine, and confirm each channel with single-stain controls (HPA tissue IHC; standard IF practice). Choose fluorophores after measuring tissue autofluorescence, favoring a cleaner spectral channel for the weaker signal (standard IF practice). REG3G is secreted and cytoplasmic with no transmembrane segment; use permeabilisation when testing an intracellular epitope, but evaluate extracellular signal without assuming membrane access is required (UniProt Q6UW15 subcellular location and topology; standard IF practice).

How do I distinguish REG3G staining from chromogenic background?

Run a no-primary control and inspect whether pigment persists in the same structures as the apparent REG3G signal (standard IHC practice). For peroxidase detection, include an endogenous peroxidase block and check background before developing DAB; these are general chromogenic workflow steps, not REG3G-specific evidence (standard IHC practice). The selected image used 3% BSA for 0.5 hour and a biotinylated secondary, so a secondary-only control can help assess nonspecific secondary or detection-system signal (caption M05473; standard IHC practice). Compare background with a positive control section under matched exposure and development conditions before increasing primary concentration (standard IHC practice).

What is a defensible way to quantify REG3G IHC? ⚠ ANSWER MARKED FOR VERIFICATION

Define tissue regions and cell classes before scoring, then apply the same rules to every slide (standard IHC practice). For cellular staining, report the percentage of positive cells and an H-score that combines staining intensity with the fraction of cells at each intensity (standard IHC practice). For extracellular or luminal REG3G, report positive area or optical density per mm² of the relevant compartment rather than assigning that signal to nearby cells (UniProt Q6UW15 secretion; standard IHC quantification). Normalize to the sampled compartment area or eligible cell count, and record section quality and background thresholds alongside results (standard IHC practice).

What findings would make a REG3G-positive result doubtful?

A crisp membrane-only pattern deserves scrutiny because REG3G has no annotated transmembrane segment and is classified as secreted and cytoplasmic (UniProt Q6UW15 topology and subcellular location). Compare staining with the selected human pancreas image and with HPA’s Paneth-cell observations, while recognizing that HPA calls its tissue IHC reliability uncertain (caption M05473; HPA tissue IHC and reliability). Signal confined to section edges, necrotic areas, or sites also positive in no-primary controls favors artefact (standard IHC practice). Residual endogenous enzyme activity can mimic DAB staining; confirm a proposed positive with appropriate blocking and an independently supported tissue pattern (standard IHC practice).

Boster reagents

Best REG3G / Regenerating islet-derived protein 3-gamma IHC Antibodies

M05473 is a human-reactive REG3G antibody with real IHC-P staining data from paraffin-embedded human pancreas sections (M05473 catalog reactivity; M05473 IHC image caption).

Real IHC data

Anti-REG3G Antibody

Cat # M05473

View product →

M05473 is listed for human IHC-P (M05473 catalog applications and reactivity). Its IHC image caption reports REG3G staining in formaldehyde-fixed, paraffin-embedded human pancreas sections (M05473 IHC image caption).

Which to pick: For tissue IHC, choose M05473, a mouse monoclonal listed for human IHC-P at 1:25 (M05473 catalog host, clone, applications, reactivity, and dilution). Its own image caption documents staining in formaldehyde-fixed, paraffin-embedded human pancreas sections (M05473 IHC image caption). There is no supported IF/ICC or cross-species pick in this payload (M05473 catalog applications, reactivity, and IF image data).

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

References

UniProt Consortium. UniProt entry Q6UW15 (REG3G_HUMAN, Regenerating islet-derived protein 3-gamma).
Human Protein Atlas. REG3G tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. REG3G subcellular location (ICC-IF): Localized to the cytosol. Caution: Based on antibodies targeting proteins from multiple genes..
Human Protein Atlas. REG3G antibody validation summary (3 antibodies).
Intestinal REG3 Lectins Protect against Alcoholic Steatohepatitis by Reducing Mucosa-Associated Microbiota and Preventing Bacterial Translocation. Cell host & microbe 2016 — PMC4786170.
The colonic macrophage transcription factor RBP-J orchestrates intestinal immunity against bacterial pathogens. The Journal of experimental medicine 2020 — PMC7144519.
Acceleration of pancreatic tumorigenesis under immunosuppressive microenvironment induced by Reg3g overexpression. Cell death & disease 2017 — PMC5636971.
P. gingivalis Lipopolysaccharide Stimulates the Upregulated Expression of the Pancreatic Cancer-Related Genes Regenerating Islet-Derived 3 A/G in Mouse Pancreas. International journal of molecular sciences 2020 — PMC7583020.
PubMed PMID:15556304 — UniProt-cited evidence.
PubMed PMID:15777617 — UniProt-cited evidence.
PubMed PMID:12975309 — UniProt-cited evidence.