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INSL3 / Insulin-like 3 · IHC design guide
Design Immunohistochemistry for INSL3
Use testicular Leydig-cell cytoplasmic staining as the INSL3 IHC reference (HPA tissue IHC). This guide covers paraffin-section handling, controls and the catalog antibody’s 1:50–1:100 IHC-P dilution (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
INSL3 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 | Leydig-cell cytoplasm in testis (HPA tissue IHC) | |
| Staining pattern | Selective cytoplasmic staining of testicular Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01712) | |
| Caveat | Secreted protein; tissue staining may differ from local RNA (HPA tissue IHC) | |
| Regulation | Prenatal and postnatal Leydig expression (UniProt) | |
| Isoform / epitope | 2 isoforms; processing may alter epitopes, but the epitope is unmapped (UniProt) |
Section 1
Recommended INSL3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by three published INSL3 IHC methods (PMC8382946; PMC12420327; PMC8072358).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A01712); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| 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-INSL3, 1:50 - 1:100 (datasheet A01712) |
| 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 | INSL3-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in Leydig cells in testis. No signal in the no-primary control. |
💡Decision noteTry citrate pH 6.0 heat retrieval first (page retrieval rule; PMC8382946: methods), then optimize for the tissue and fixative used.
Section 2
What Is the Expected INSL3 Staining Pattern?
INSL3 is secreted and has no transmembrane segment (UniProt P51460: topology). In IHC, expect selective cytoplasmic staining of testicular Leydig cells (HPA: Enhanced; High in Leydig cells).
| Strong Leydig-cell cytoplasmic stain (HPA: High in Leydig cells) | Expected pattern (HPA: selective cytoplasmic Leydig-cell expression). |
| Nuclear-only signal (HPA: cytoplasmic Leydig-cell pattern) | Discordant localisation; assess artefact (HPA: cytoplasmic Leydig-cell pattern). |
| Stain in non-Leydig cells (HPA: selective Leydig-cell pattern) | Check cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse stain across the section (HPA: selective Leydig-cell pattern) | Possible background; compare a no-primary control (general IHC practice). |
| No stain in testicular Leydig cells (HPA: High in Leydig cells) | Unexpected; check the positive control and assay steps (HPA: High; general IHC practice). |
💡Expected INSL3 appearanceCall positive for High Leydig-cell cytoplasmic stain; nuclear-only or widespread stain is suspect (HPA: tissue IHC).
| Secreted protein (UniProt P51460: subcellular location) | RNA and protein tissue locations may differ (HPA: reliability description). |
| Precursor processing (UniProt P51460: signal 1–20; propeptide 58–104) | Interpret detection in light of the antibody's epitope, if known (UniProt P51460: processing). |
| Two isoforms (UniProt P51460: isoforms 1, 2) | Check isoform coverage if the antibody epitope is known (UniProt P51460: isoforms 1, 2). |
| IF/ICC? (HPA: subcellular record) | No HPA ICC-IF images or main location are available; validate IF separately (HPA: ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| No Leydig-cell signal (HPA: High in Leydig cells) | Assay sensitivity or reagent failure is possible (general IHC practice). | Check positive control, retrieval, dilution and detection (general IHC practice). |
| Nuclear-only stain (HPA: cytoplasmic Leydig-cell pattern) | Localisation is discordant (HPA: cytoplasmic Leydig-cell pattern). | Review morphology and no-primary control (general IHC practice). |
| Non-Leydig cells stain (HPA: selective Leydig-cell pattern) | Cross-reactivity or endogenous activity is possible (general IHC practice). | Compare no-primary and negative-tissue controls (general IHC practice). |
| Diffuse section-wide stain (HPA: selective Leydig-cell pattern) | Background from detection or inadequate blocking is possible (general IHC practice). | Check no-primary control; optimise blocking and washes (general IHC practice). |
| Adipocytes stain in adipose tissue (HPA: Not detected) | Unexpected signal may be nonspecific (HPA: Not detected; general IHC practice). | Compare a no-primary control and reassess scoring (general IHC practice). |
Experiment evidence
IHC & IF images showing INSL3 staining
A01712 · IHCImmunohistochemistry analysis of paraffinembedded Human prostate using INSL3 pAb at dilution of 1:100 (40xlens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/cerebral+cortex#imid_7109920
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/colon#imid_7109838
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/liver#imid_7109839
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/kidney#imid_7109832
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for INSL3 IHC & IF
🧪Run testis first: Leydig cells should stain (HPA: High in testis Leydig cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); non-Leydig cells on the testis slide should provide an internal background comparison (HPA: High staining is assigned to Leydig cells).
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for INSL3; derive a cell-line control from the positive tissue's cell type (Leydig cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a host-species-matched nonimmune IgG control for the polyclonal catalog antibody (standard IHC practice; selected-SKU caption: pAb). Use an INSL3 knockout specimen, if available, as a biological specificity control; check testis sections for endogenous peroxidase signal in chromogenic IHC and autofluorescence in IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and any fixation effect are unreported in the supplied evidence; the selected A01712 paraffin-section caption does not state a fixative (selected-SKU caption). Retrieval dependence and whether frozen sections or IF are easier than paraffin IHC are also unreported (supplied target/application evidence). No testis-specific artefact is documented here; assess background with the controls above (supplied evidence; standard IHC/IF practice).
HPA tissue IHC evidence for INSL3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced INSL3 IHC Tips
Troubleshoot paraffin-section INSL3 IHC against Leydig-cell staining, while accounting for secretion, processing, and the limits of the available antibody evidence.
Which retrieval should I start with for paraffin INSL3 IHC, and how should I assess weak staining?
Start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval setting). The A01712 paraffin-section caption reports 1:100 antibody dilution but does not report retrieval conditions (caption A01712). Compare serial testis sections containing Leydig cells, keeping section thickness and chromogen development consistent (HPA: high staining in Leydig cells; standard IHC practice). If staining is weak, test EDTA pH 9.0 on adjacent sections and assess morphology before changing antibody concentration (standard IHC practice). Look for selective cytoplasmic staining, since INSL3 is secreted and has no transmembrane segment (HPA: Leydig-cell cytoplasm; UniProt P51460: localisation and topology).
Could fixation explain weak or uneven INSL3 staining in paraffin sections?
Target-specific sensitivity to fixation is unknown; the A01712 image identifies paraffin sections but gives no fixative or fixation time (caption A01712). For troubleshooting, compare specimens with documented fixation histories and run them through the same retrieval and chromogen steps (standard IHC practice). Where preparation can be controlled, use a consistent 10% neutral buffered formalin workflow and record specimen thickness and fixation duration (standard IHC practice). Inspect paired sections for uneven morphology and staining before attributing a difference to INSL3 abundance (standard IHC practice). Use testis Leydig cells as a staining reference, while treating their expected pattern as localisation evidence rather than evidence about fixation tolerance (HPA: selective Leydig-cell cytoplasm).
What staining pattern should I expect, and how should I assess signal outside Leydig cells?
Expect selective cytoplasmic staining in testis Leydig cells when assessing a positive tissue control (HPA: enhanced tissue IHC, selective Leydig-cell cytoplasm). INSL3 is secreted and has no transmembrane segment, so a crisp plasma-membrane outline is not the expected cellular pattern (UniProt P51460: localisation and topology). Review surrounding tissue for diffuse extracellular color, comparing its distribution with intact Leydig-cell cytoplasm and section morphology (standard IHC practice; HPA: Leydig-cell cytoplasm). The A01712 image documents staining in a human prostate paraffin section at 1:100, without establishing a Leydig-cell pattern there (caption A01712). Include a no-primary control to assess deposition unrelated to primary-antibody binding (standard IHC practice).
How could INSL3 processing or isoforms affect what my IHC antibody detects?
INSL3 has 2 listed isoforms and a precursor with a signal peptide at residues 1–20 and propeptide at 58–104 (UniProt P51460: isoforms and processing). Its mature hormone is described as disulfide-linked A and B chains, making epitope placement relevant to interpretation (UniProt P51460: subunit). Check the antibody’s documented immunogen or epitope against these regions before interpreting a negative result as absence of all INSL3 forms (standard IHC practice; UniProt P51460: processing). No glycosylation sites or modified residues are listed, so this record supplies no basis for a glycosylation-specific staining explanation (UniProt P51460: PTMs). Compare staining across the same control tissue under matched retrieval conditions (standard IHC practice).
How should I investigate INSL3 by IF when I need to multiplex cell identity?
On the separate IF/ICC guide, pair INSL3 with a validated Leydig-cell marker to test whether signal occupies the expected cells (HPA: selective Leydig-cell cytoplasm; standard IF practice). Select spectrally separated fluorophores, favoring a far-red channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). Include single-label and no-primary controls to distinguish bleed-through and autofluorescence from antibody signal (standard IF practice). For an intracellular epitope, compare mild permeabilisation with an unpermeabilised condition; interpret accessible extracellular signal separately because INSL3 is secreted (UniProt P51460: localisation; standard IF practice). The supplied HPA subcellular record lists no ICC/IF images, so it does not establish an IF staining pattern for this antibody (HPA: subcellular record).
How can I separate genuine INSL3 signal from diffuse chromogenic background?
First inspect a no-primary section and a matched positive testis section to locate nonspecific color relative to Leydig-cell cytoplasm (standard IHC practice; HPA: Leydig-cell cytoplasm). For a peroxidase-based assay, check the hydrogen-peroxide block before DAB development, especially where endogenous enzyme activity may contribute color (standard IHC practice). Check blocking, washes, and chromogen development time under identical conditions, then titrate antibody if diffuse staining persists (standard IHC practice). The A01712 caption reports 1:100 for a human prostate paraffin image; treat that as an image condition requiring validation in your material (caption A01712). Diffuse color across unrelated cell types is less persuasive than selective Leydig-cell cytoplasm (HPA: selective Leydig-cell expression).
What should I quantify when comparing INSL3 IHC across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score cytoplasmic staining within identified Leydig cells, reporting the percentage positive and intensity for each specimen (HPA: selective Leydig-cell cytoplasm; standard IHC practice). An H-score can combine intensity grades 0–3 with their cell percentages, yielding a 0–300 scale (standard IHC practice). Define the same Leydig-cell identification rule, chromogen exposure, and analysis threshold before comparing slides (standard IHC practice). Normalise positive-cell counts to the number of Leydig cells assessed, or stained area to the annotated Leydig-cell compartment, and report the denominator (standard IHC practice). Record section quality and exclude folds, edges, and necrotic regions according to a prespecified rule (standard IHC practice).
When should I question an apparent INSL3-positive IHC result?
A convincing positive pattern includes cytoplasmic staining in morphologically identified testis Leydig cells (HPA: enhanced IHC, selective Leydig-cell cytoplasm). Question a predominantly nuclear or sharply membrane-restricted pattern, since INSL3 is secreted and has no transmembrane segment (UniProt P51460: localisation and topology). Compare staining at cut edges, folds, and necrotic areas with intact tissue, and review a no-primary control for chromogen or endogenous-enzyme signal (standard IHC practice). Widespread staining in unrelated cell populations needs additional antibody-specific controls before being called INSL3 (HPA: selective Leydig-cell expression; standard IHC practice). The A01712 prostate paraffin image demonstrates an image condition at 1:100, not specificity across tissues (caption A01712).
Boster reagents
Best INSL3 / Insulin-like 3 IHC Antibodies
A01712 has a real IHC image from a human prostate paraffin section and is listed for IHC-P and IF/ICC in human, mouse and rat (image caption; catalog: applications, reactivity).
Real IHC data
Anti-Insulin-like 3 INSL3 Antibody
Cat # A01712
A01712 will render with an IHC image of human prostate paraffin tissue stained at 1:100 (image caption). It is listed for IHC-P and IF/ICC in human, mouse and rat; no IF image is supplied (catalog: applications, reactivity, IF image alts).
Which to pick: Choose A01712 for paraffin-section tissue IHC because its own image shows staining in human prostate at 1:100; the fixative is unreported (image caption). A01712 is also listed for IF/ICC and for human, mouse and rat reactivity, although the payload supplies no IF image (catalog: applications, reactivity, IF image alts). It has a rabbit host, and its clone is unspecified (catalog: host, clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P51460 (INSL3_HUMAN, Insulin-like 3).
- Human Protein Atlas. INSL3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. INSL3 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. INSL3 antibody validation summary (1 antibodies).
- Serum Concentrations and Gonadal Expression of INSL3 in Eighteen Males With 45,X/46,XY Mosaicism. Frontiers in endocrinology 2021 — PMC8382946.
- Insulin-like peptide 3 is not a biomarker for pancreatic ductal adenocarcinoma. Frontiers in endocrinology 2025 — PMC12420327.
- Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells. International journal of environmental research and public health 2016 — PMC4808909.
- The Production of Testosterone and Gene Expression in Neonatal Testes of Rats Exposed to Diisoheptyl Phthalate During Pregnancy is Inhibited. Frontiers in pharmacology 2021 — PMC8072358.
- PubMed PMID:8034302 — UniProt-cited evidence.
- PubMed PMID:8020942 — UniProt-cited evidence.
- PubMed PMID:7852540 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for INSL3 (Insulin-like 3). Expected localisation Leydig-cell cytoplasm in testis (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A01712 at 1:50 - 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A01712); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-INSL3, 1:50 - 1:100 (datasheet A01712); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01712); Caveat: Secreted protein; tissue staining may differ from local RNA (HPA tissue IHC); Isoform / epitope: 2 isoforms; processing may alter epitopes, but the epitope is unmapped (UniProt); Regulation: Prenatal and postnatal Leydig expression (UniProt). Evidence: UniProt P51460, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A01712, PMC8382946, PMC12420327. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/INSL3/immunohistochemistry-ihc-guide.